Rozprawy doktorskie: „Vaccins ADN”

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Marsac, Delphine. "Utilisation de vaccins ADN codant pour des pseudoparticules virales comme outils de présentation d'antigènes du VIH-1 et du VIS pour l'induction d'une réponse immunitaire T in vivo". Paris 5, 2004. http://www.theses.fr/2004PA05N03S.

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La présentation exogène de particules VIH-1, restreinte par les molécules de classe 1 du CMH active des CTL spécifiques. Nous avons étudié l'immunogénicité de particules Gag du VIH-1 pseudotypées par la protéine G d'enveloppe du virus de la stomatite vésiculeuse produite par vaccination génétique chez la souris. L'enveloppe VSV-G en facilitant l'entrée des particules virales dans la cellule, augmente l'efficacité d'induction de CTL anti-Gag en favorisant la présentation d'épitopes Gag par les molécules du CMH de classe I et II. L'étude de l'immunogénicité de vecteurs ADN codant pour des pseudoparticules formées par les protéines d'enveloppe du virus de l'hépatite B présentant des domaines antigéniques du VIH-1 et du virus de l'immunodéficience simienne a montré l'induction de réponses T spécifiques in vivo. Après rappel par un virus
Major histocompatibility complex class I exogenous presentation of HIV-1 particules activates specific CTL responses. We used DNA-based immunization with plasmids codingfor HIV-1 Gag particles pseudotyped with vesicular stomatitis virus glycoprotein. The presence of the VSV-G enveloppe increased the efficiency of the specific anti-Gag lysis due to a better presentation of the Gag epitopes by MHC class I and II processing pathway. We also improved the immunogenicity of DNA vaccines encoding hepatitis B surface antigen fused to antigenic domains of simian/human immunodeficiency viruses in mice and macaques rhesus. Immunization with hybrid DNA induced effector and long-lasting precursors T, cells, efficiently

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Hechard, Céline. "Vaccination ADN contre la chlamydiose abortive ovine : évaluation de la protection des vaccins ADN MOMP, DnaK et GroEL dans un modèle murin d'infection". Tours, 2002. http://www.theses.fr/2002TOUR4002.

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Chlamydophila abortus est une bactérie intracellulaire obligatoire qui infecte principalement les brebis mais peut-être également transmise à l'homme. Elle est responsable d'avortements tardifs sans signe avant-coureur ou de la mise bas de petits chétifs, difficiles à élever. Les avortements ovins occasionnent des pertes économiques importantes dans les élevages. Dans une stratégie de vaccination ADN, les séquences codant pour des protéines immunogènes de C. Abortus ont été clonées dans le vecteur d'expression eucaryote pcDNA3. 1. L'effet protecteur des vaccins ADN contenant les séquences codant pour la protéine majeure de la membrane externe (MOMP) et les protéines de choc thermique, DnaK et GroEL ont été évalués dans un modèle murin de gestation. Les injections intramusculaires des ADN vaccinaux ont protégé partiellement les souris des avortements induits par l'infection. La charge bactérienne des rates des souris gestantes ou non, et des placentas n'a pas été réduite par la vaccination ADN. Toutefois, nous avons observé une protection fœtale probablement due à l'effet barrière du placenta. La réponse humorale générée par la vaccination ADN s'est avérée spécifique mais faible. Elle implique des anticorps, d'isotype majoritaire IgG2a, mais qui n'ont pas de pouvoir neutralisant in vitro sur l'infectivité de C. Abortus. Afin d'optimiser la réponse immunitaire induite par la vaccination ADN avec le gène codant pour la protéine DnaK, un rappel protéique a été réalisé avec la protéine DnaK exprimée chez E. Coli. Bien que cette stratégie ait considérablement augmentée la réponse humorale, aucune modification de la protection induite par le vaccin ADN seul n'a été obtenue. Il semblerait donc que, dans ce cas, l'antigène ait un faible pouvoir protecteur. Compte tenu que les antigènes immunogènes connus pour être protecteurs dans d'autres modèles n'ont donné que des résultats modérés en vaccination ADN contre C. Abortus, nos prochains travaux consisteraient à identifier de nouveaux antigènes protecteurs en vaccination ADN. Dans ce but, nous pourrions utiliser une nouvelle stratégie prometteuse, consistant au criblage d'une banque génomique d'ADN bactérien, en fonction de son pouvoir protecteur in vivo, après immunisation génique.

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Robin, Marie. "Vaccination ADN dans la leucémie aiguë promyélocytaire et étude de la réponse immune". Paris 7, 2007. http://www.theses.fr/2007PA077073.

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La leucémie aiguë promylocytaire (LAP) représente 10% de l'ensemble des leucémies aiguës promyélocytaires et se caractérise par la translocation chromosomique (15 ; 17), à l'origine d'un transcrit de fusion associant PML et le récepteur alpha à l'acide rétinoïque (RARalpha). Le traitement par l'acide tout trans-rétinoïque (ATRA) et la chimiothérapie permet d'obtenir une rémission chez plus de 90% des patients par une différentiation des cellules leucémiques induite par l'ATRA. Malheureusement, 10 à 30% des patients rechutent. Le but de notre travail est de tester un vaccin anti-leucémique et d'étudier l'immunogénicité spécifique dans cette maladie. Dans un modèle murin de LAP, nous avons mis en place un protocole de vaccination anti-leucémique par un plasmide composé d'un promoteur, d'un adjuvant (fragment Fc de la toxine tétanique) et d'un gène codant pour la fusion protéique PML-RAR. Dans ce modèle, les souris vaccinées avaient un avantage de survie, en particulier lorsque le traitement associait vaccin et ATRA. Les mécanismes d'action de cette immunothérapie allient réponse cellulaire et humorale comme montré par l'augmentation de sécrétion de l'interféron, la lyse cellulaire spécifique des cibles LAP et l'apparition d'anticorps spécifiques contre RARalpha. Dans cette première étude, nous avons constaté que la production d'anticorps augmentait au cours du temps et que la présence d'anticorps à J18 était corrélée à la survie. Chez l'homme traité selon les protocoles usuels, nous avons également observé une sécrétion d'anticorps, qui dans la moitié des cas, étaient déja�� présents au diagnostic à un faible taux. Sur 9 patients testés, les taux d'anticorps, comme dans le modèle murin, étaient croissants au cours du temps, suggérant que le traitement s'accompagne d'un accroissement du taux d'anticorps. Par ailleurs, les patients avaient également dans plus de 50% des cas soit des anticorps anti-nucléaires, soit des anticorps anti-cytoplasme de neutrophiles. En conclusion, le vaccin ADN PML RAR est une immunothérapie encourageante, notamment chez les patients à haut risque de rechute, au moment où la charge leucémique faible. La présence d'anticorps anti-RARalpha doit continuer d'être évaluée dans les essais prospectifs en cours afin d'établir s'il existe une corrélation entre le taux d'anticorps et les paramètres cliniques et biologiques du malade
Acute promyelocytic leukemia (APL) represents 10% of all acute myeloblastic leukaemia and is characterized by a reciprocal chromosomal translocation between 15 and 17 fusing PML and the retinoid acid receptor alpha (RARalpha). Treatment with all trans-retinoid acid (ATRA) and chemotherapy induce complete remission in more than 90% of patients with APL through ATRA-induced differentiation of the leukemic cells. Unfortunately, 10 to 30% of patients relapse. The aim of our work was to test a DNA antileukemic vaccine coding for the PML-RARalpha jonction and study the specific immunogenecity in this disease. In an APL transplantable mice model, we set up a vaccine protocol using a plasmid coding for a promoter, an adjuvant (Fc fragment from tetanie toxin) and fusion protein PML-RAR. In this model, vaccinated mice had a better survival, in particular when they received vaccine and ATRA. Mechanism of for this immune response was cellular and humoral response as demonstrated by increased interferon secretion, specific APL target lysis and specific antibodies against RARalpha. In this first study, we observed that antibody production increased with time and that antibody production on day 18 was predictive for a better survival. In humans treated according to usual protocols, we also observed an antibody secretions which in half of patients, were already present at low level at diagnosis. In 9 tested patients, antibody production was increasing with time as in mice, suggesting maintenance treatment is associated with an increased production of antibody. Furthermore, 50% of patients had also anti-nuclear and anti-neutrophil cytoplasmic antibody. We conclude that DNA vaccine is an encouraging targeted immunotherapy, in particular in patients at high risk of relapse, when patients had low leukemic burden. Antibody production should continue to be evaluated through ongoing prospective immunomonitoring of patients with APL from diagnosis throughout treatment to establish whether there is a correlation between anti-RARalpha and clinical or other biological parameter

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Rolland-Turner, Magali. "Développement d'un vaccin immunocontraceptif : mise au point de tests immunologiques dans le modèle vulpin et développement de vaccins ADN avec les antigènes spermatiques fSP13 et fSP8". Nancy 1, 2005. http://www.theses.fr/2005NAN11303.

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Le but de ce travail était tout d'abord, la mise au point d'outils immunologiques permettant l'analyse de la réponse immune humorale et cellulaire chez le renard (Vulpes vulpes). Après avoir sélectionné des anticorps reconnaissant les différentes classes d'immunoglobuline de renard, le modèle antigénique ovalbumine et choléra toxine B a été utilisé pour mettre au point les techniques ELISA et ELISPOT. Quatre cytokines vulpines Il2, Il6, Il10 et INFy ont été clonées et séquencées, et une technique d'analyse de leur expression par RT-PCR quantitative, après re-stimulation antigénique in vitro de PBMCs vulpins a été mise au point. Par la suite, différents vaccins ADN utilisant le vecteur vaccinaI commercial pVAX1 et les antigènes spermatiques spécifiques du testicule fSP13 et fSP8 identifiés au laboratoire ont été construits. La fonctionnalité de celles-ci à tout d'abord été testée in vitro pour l'expression de fSP 13 et fSP8 après transfection de cellules MDCK.

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Faurez, Florence. "Plasmide vaccinal réplicatif chez le porc : biosécurité". Rennes 1, 2010. http://www.theses.fr/2010REN1S023.

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Plusieurs stratégies d’amélioration du vaccin ADN ont été étudiées cependant peu d’études sur la biosécurité des ces nouvelles stratégies ont été réalisées. Ce projet de thèse apporte des réponses sur l’étude de biosécurité sur un plasmide vaccinal rendu réplicatif à partir d’éléments viraux du Circovirus porcin de type 2 (PCV2). L’évaluation de la biosécurité d’un plasmide réplicatif dérivé d’éléments viraux comprend différents paramètres tels que sa caractérisation in vitro, l’évaluation de son efficacité au niveau vaccinal, sa distribution dans l’organisme, la caractérisation de la réplication de l’ADN, sa cinétique d’élimination et son nombre d’évènements d’intégration dans le génome de l’organisme. Afin de contribuer à l’évaluation de la biosécurité d’un plasmide réplicatif, utilisé en tant que plasmide vaccinal, nous avons apporté un panel de plasmides réplicatifs dérivés de PCV2, une méthode de détermination du taux de réplication de plasmides réplicatifs et la validation du protocole utilisé dans l’étude de distribution des plasmides chez le porc
Several strategies to improve DNA vaccine have been studied but few studies on the biosafety of these new strategies were carried out. This thesis provides some answers on biosafety of a replicative plasmid derived from replicative elements of porcine circovirus type 2 (PCV2). The biosafety assessment of a replicative plasmid derived from viral elements include parameters such as its characterization in vitro, assessment of its effectiveness in a vaccine, its distribution in the body, the characterization of the replication of DNA, its kinetics of elimination and number of events integrated into the genome of the organism. To contribute in part to assess the biosafety of replicative plasmid, used as a plasmid vaccine, we made a panel of replicative plasmids derived from PCV2, a method determining the rate of replication of replicative plasmids and the validation protocol used in the study of distribution of plasmids in pigs

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Desolme, Benoît. "Vaccination par ADN contre la toxoplasmose : application au modèle murin avec les gènes GRA4 et SAG1 de Toxoplasma gondii : stratégies d'optimisation de la protection". Tours, 1999. http://www.theses.fr/1999TOUR3804.

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Innocentin, Silvia. "Utilisation de bactéries lactiques recombinantes invasives comme outil novateur pour la vaccination ADN par voie muqueuse". Versailles-St Quentin en Yvelines, 2008. http://www.theses.fr/2008VERS0012.

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Nous avons étudié la possibilité d’utiliser les Bactéries Lactiques (BL) pour vectoriser des vaccins ADN par voie muqueuse. Des BL alimentaires avaient déjà été utilisées pour délivrer des protéines par cette voie. Nous avons montré que la BL modèle Lactococcus lactis peut délivrer un vecteur plasmidique d’expression eucaryote codant pour un allergène majeur du lait de vache, la beta-lactoglobuline (BLG) dans la lignée Caco-2 et permettre l’expression de la protéine BLG par les cellules. Pour améliorer l’efficacité du transfert d’ADN, nous avons utilisé des souches de L. Lactis rendues invasives par expression des gènes InlA de Listeria monocytogenes et FnBPA de Staphylococcus aureus. Ces deux types de souches présentent des taux d’internalisation similaires et la même capacité à vectoriser un plasmide d’expression de la GFP (1% des cellules Caco-2 exprimant la GFP). Nous avons ensuite testé les lactocoques invasifs comme vecteurs d’ADN vaccinant dans deux modèles pathologiques chez la souris : l’allergie à la BLG et l’infection par le virus de la grippe. In vitro, les cellules Caco-2 exprimaient 30 fois plus de BLG après co-incubation avec les souches invasives comparées aux souches non-invasives. In vivo, l’administration intranasale de souches invasives FnBPA+ et de souches non-invasives induit respectivement une réponse immunitaire Th2 et Th1 contre la BLG. L’administration intranasale de souches invasives FnBPA+ conçues pour permettre l’expression de l’hémagglutinine et de la nucléoprotéine du virus de la grippe se sont révélées moins efficaces que la vaccination génétique classique (ADN nu par voie intradermique) pour protéger les souris contre une épreuve
In this study, we evaluate the potential of Lactic Acid Bacteria (LAB) as mucosal DNA vaccine delivery vectors. LAB are food-grade bacteria already used to deliver proteins at the mucosal level. We showed that Lactococcus lactis, a model LAB, can deliver a eukaryotic expression plasmid coding for a major cow’s milk allergen, beta-lactoglobulin (BLG) gene in Caco-2 cell line with subsequent expression of BLG protein by the cells. To improve DNA delivery, we used L. Lactis strains rendered invasive by expressing Listeria monocytogenes InlA or Staphylococcus aureus FnBPA genes. Both showed comparable internalization rates and ability to deliver a GFP expression plasmid: 1% of Caco-2 cells expressed GFP while no GFP was detected with non invasive strains. We then tested invasive L. Lactis as DNA vaccine carrier in two disease models of mouse: allergic response to BLG and influenza virus infection. In vitro, Caco-2 cells expressed 30-fold more BLG when co-incubated with invasive strains compared to non invasive. In vivo, intranasal administration of invasive FnBPA+ strain and non invasive strains induced a Th2 and Th1 immune responses against BLG, respectively. Intranasal administration of invasive FnBPA+ strains designed to express hemagglutinin and nucleoprotein of influenza virus was less efficient than intradermal naked DNA immunization to protect mice from viral challenge

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Moussa, Maha. "Immunité et protection induites par un lentivecteur ADN innovant chez les modèles animaux de vaccination VIH-1". Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV029/document.

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Nous avons récemment développé un prototype lentivecteur ADN non intégratif vaccinal contre VIH-1/SIDA que nous avons testé chez des modèles animaux. L'immunisation avec une dose unique de ce vaccin (CAL-SHIV-IN-) a permis la mise en place rapide de réponses immunes spécifiques contre tous les antigènes exprimés par le vaccin chez tous les animaux vaccinés. Les analyses longitudinales ont démontré la mise en place de réponses cellulaires et humorales spécifiques et persistantes sur une durée de plus de 74 semaines en absence de réintroduction d'antigènes chez tous les macaques vaccinés. La caractérisation de ces réponses a révélé la présence de cellules T CD4+ et CD8+ polyfonctionnelles composées de fractions de cellules effectrices mémoires à fonction immédiate (EM), de cellules centrales mémoires (CM) et de cellules précurseurs mémoires ayant une haute capacité de prolifération (PHPC). Ces réponses corrèlent, chez tous les macaques vaccinés (6/6), avec un contrôle du virus d'épreuve hautement hétérologue et pathogénique (SIVmac251) inoculé à petites doses répétées par la voie mucosale rectale. Cette protection est maintenue durant toute la période d'un an de suivi après l'infection avec une différence statistiquement significative de la charge virale plasmatique des groupes contrôles et vaccinés au moins jusqu'à 18 semaines post-infection. Par ailleurs, le contrôle du virus d'épreuve est maintenu plus de 10 mois (correspondant au temps d'arrêt de l'étude) après l'infection. Parmi les corrélats immunologiques de protection nous avons identifié la présence de cellules de type PHPC spécifiques des antigènes du vaccin et qui sont dotées d'une capacité importante de prolifération ex vivo en présence des signaux antigéniques et homéostatiques. Nous avons démontré que ces PHPC contiennent une fraction de cellules T souches mémoires « TSCM » spécifiques du vaccin. Ces TSCM récemment identifiées constitueraient un atout majeur en faveur de notre vecteur et notre stratégie vaccinale du fait de leur haute capacité d'auto-régénération/maintien en absence d'antigène et leur capacité à se différencier en d'autres cellules mémoires TCM et TEM
We recently developed an innovative prototype non-integrative lentivector DNA vaccine against HIV-1 /AIDS that we tested in pilot studies using animal models of HIV vaccine. We found that a single immunization with our prototype vaccine (CAL-SHIV-IN-) allowed the implementation of potent humoral and cellular responses in all immunized macaques. In addition, both types of responses persisted over a period of 74 weeks post-immunization in absence of antigenic boost. The characterization of the above revealed that vaccine specific T cell responses included polyfunctional CD4+ and CD8+ T cells against all antigens expressed by the vaccine. Detailed phenotypic and functional examinations of these cells showed that they were composed of effector (EM) and central memory (CM) T cells. More importantly they also contained a fraction of precursor memory T cells with high proliferative capacity (PHPC). Immune responses primed by our vaccine regiment correlated with protection in all vaccinated macaques (6/6). As expected our vaccine-induced immune responses did not prevent from infection acquisition but controlled the replication of the highly pathogenic and heterologous SIVmac251 challenge given as repeated low dose by the intrarectal mucosal route. All vaccinated animals (6/6) controlled their viremia to undetectable level using conventional PCR during at least 10 months post infection (end of the experiment). We further focused on PHPC responses associated with viral control and found that these cells vigorously proliferate upon ex vivo stimulation with specific antigens in presence of the homeostatic IL-7 and IL-15 cytokines. Proliferating antigen specific cells contained a type of stem cell-like memory T cells (TSCM). These latter (TSCM) might be a major asset in favor of our lentivector and vaccination strategy due to their high capacity for self-regeneration/maintenance in absence of antigen source

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Szelechowski, Marion. "Vers une réplication controlée des vecteurs dérivés de l'adénovirus canin de type 2". Paris 7, 2008. http://www.theses.fr/2008PA077186.

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Parmi les vecteurs viraux développés pour la vaccination, les vecteurs dérivés des adénovirus se sont avéré prometteurs. Des vecteurs réplicatifs et défectifs d'un sérotype canin d'adénovirus, Cav2, ont été dérivés et nous avons pu démontrer leur efficacité à mettre en place une immunité protectrice contre le produit du transgène chez la souris puis chez le mouton. L'objectif de ce travail de thèse est de proposer une alternative à l'utilisation de ces deux types courants de vecteurs en vaccination, avec le développement de vecteurs semi-réplicatifs dérivés de Cav2. Ces vecteurs doivent permettre la réplication du génome viral sans production de nouvelles particules infectieuses, et posséder ainsi à la fois les caractéristiques d'efficacité des vecteurs réplicatifs et de sécurité des vecteurs défectifs. Ces propriétés peuvent être obtenues par la délétion sur le génome viral de séquences codant des protéines tardives de Cav2 indispensables à la formation de particules infectieuses mais non nécessaires à la réplication du génome viral. Afin d'être en mesure de manipuler précisément le génome viral, nous avons réalisé une cartographie complète de l'unité de transcription tardive de Cav2. Nous avons ensuite réalisé des délétions totales ou partielles des cadres de lecture correspondant à nos cibles, ou inséré des mutations non-sens, par recombinaison homologue sur le génome sauvage. Pour la production des vecteurs, nous avons dû construire des lignées cellulaires de complémentation, apportant en trans les protéines cibles respectives. Enfin, nous avons pu vérifier in vitro les caractéristiques semi-réplicatives de vecteurs Cav2 délétés de la fonction protéase
Among the vectors derived for vaccination purpose, those derived from adenoviruses revealed particularly hopeful results. Replicative and defective vectors have been developed from Cav2, and we were able to demonstrate their to settle a protective immune response against the transgene product both in mice and sheep. This work aims to propose an alternative vector for vaccination derived from Cav2 and characterized by an abortive or semi-replicative behavior in the transduced cells. This vector should enable the genomic replication without production of new infectious particles. It should therefore possess both replicative vectors efficacy and defective vectors security. This can be achieved by the deletion of defined viral genomic regions, in particular within the late expression region which is dispensable for the first stages of the replication cycle. To manipulate accurately the viral genome, we elaborated a transcriptional map of the late transcriptional unit of Cav2 génome. Deletion of ail or part of the targeted open reading frames were then realized, or nor sens mutation were introduced within them, by homologuous recombinaison on the wild type Cav2 genome. Then we constructed complementing cell lines expressing the viral deleted proteins, in order to produce the recombinant viral particles. Finally, the semi-replicative properties of protease deleted Cav2 were confirmed by in vitro analysis

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Bernelin-Cottet, Cindy. "Développement d'un vaccin à ADN contre le virus du Syndrome Dysgénésique et Respiratoire Porcin (PRRSV)". Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLA004/document.

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Le Syndrome Dysgénésique et Respiratoire Porcin (PRRS) est la maladie infectieuse endémique la plus couteuse en élevage porcin dont l'agent responsable est un Arterivirus, le PRRSV, qui présente une grande diversité génétique. L'infection par le PRRSV est fréquemment associée à l'infection par les virus influenza. La vaccination est une méthode de lutte adaptée contre ces virus. Dans le cas du PRRSV, les vaccins les plus utilisés sont des virus vivants modifiés (MLV) qui induisent une immunité protectrice peu efficace contre les variants viraux. Dans le cas du virus influenza, les vaccins inactivés utilisés présentent la même insuffisance.Dans ce travail de thèse, j'ai évalué des stratégies vaccinales visant à induire une immunité efficace contre des variants viraux, en utilisant des antigènes conservés entre souches, adressés aux cellules présentatrices d'antigènes (APC), et j'ai analysé l'effet de différentes voies et modes d'administration.Dans le cas du virus grippal, le ciblage d'antigènes conservés (HA2, M2e, NP) au CD11c a permis d'augmenter la réponse T uniquement lors d'administration par voie intramusculaire (IM) et fut sans effet sur la réponse anticorps. La vaccination par voie intradermique s'est traduit par une exacerbation de la pathologie lors d'une épreuve virale, alors que la vaccination par voie IM a réduit les symptômes, la durée d'excrétion virale en corrélation avec une meilleure réponse anticorps anti-HA2 et M2e.Dans le cas du virus PRRSV qui fut mon sujet principal d'étude, j'ai cherché à optimiser des réponses lymphocytaires T IFNγ en employant une stratégie vaccinale ADN codant des antigènes contenant des épitopes T conservés entre souches, ciblés aux APC. En effet, alors que les mutations virales conduisent à un échappement aux anticorps neutralisants, la réponse lymphocytaire T IFNγ a été proposée impliquée dans la protection croisée. J'ai montré que l'immunogénicité optimale de vaccins ADN PRRSV, conduisant à la réponse T la plus large, est obtenue par l'administration intradermique associée aux nanoparticules de PLGA (NP), suivi d'une électroporation (EP), par rapport à EP seul ou délivrance intradermique ou transcutanée avec des patches à micro-aiguilles résorbables. Cette immunogénicité optimale est associée à une bonne transfection des cellules de la peau, à une accumulation de cellules inflammatoires, et à une mobilisation des cellules dendritiques. J'ai ensuite utilisé ce mode d'administration EP+NP pour immuniser des porcs avec des plasmides codant des antigènes conservés du PRRSV adressés ou non aux APC via CD11c ou XCR1. Les porcs ont été immunisés soit avec des injections répétées d'ADN seul soit en prime-boost ADN-MLV. Le régime ADN-MLV s'est montré supérieur pour l'induction de réponse B et T à celui de l'ADN ou du MLV seuls, et le ciblage aux APC a nettement augmenté la réponse anticorps mais pas la réponse T IFNγ. Dans une expérience suivante à visée d'application sur le terrain, j'ai utilisé le régime ADN-MLV (sans NP cette fois), délivré avec EP ou avec jet sous pression (PJ). Dans ces conditions, la primo-vaccination avec ADN n'a pas significativement augmenté la réponse T IFNγ induite par le MLV, mais elle a clairement augmenté la réponse anticorps avec un bénéfice du ciblage des APC. L'immuno-potentialisation induite par la primo-vaccination ADN n'a pas conduit à l'amélioration de la protection contre une épreuve avec un virus hétérologue et a montré que cette protection n'est au final pas corrélée avec la réponse lymphocytaire T IFNγ et opère en l'absence d'anticorps neutralisants détectables. Enfin, l'ensemble de ce travail montre que l'effet du ciblage des APC chez le porc est influencé par la voie d'administration et par le régime d'administration comme le prime-boost ADN-MLV
The Porcine Reproductive and Respiratory Syndrome (PRRS) is the most damaging infectious disease in pigs worldwide. The etiologic agent is an Arterivirus, the PRRSV, which presents a large genetic diversity. PRRSV infection is frequently associated with influenza virus co-infection. Vaccination is a highly suitable way to control these viruses. In the case of PRRSV, the most effective commercial vaccines are modified live vaccines (MLV) which induce only a partial protection against heterologous strains. In the case of the influenza virus, the available inactivated vaccines show the same weakness.With the goal to control emerging influenza and PRRSV variants, I evaluated vaccine strategies involving conserved viral antigens between strains which were targeted to antigen-presenting cells (APC) and delivered by different routes and methods.In the case of influenza virus, the targeting of conserved antigens (HA2, M2e and NP) to CD11c led to increased IFNγ T cell responses only when vaccines were delivered by the intramuscular (IM) route and had no effect on the humoral response. The intradermal route exacerbated disease following challenge whereas the IM route reduced the symptoms, the duration of viral excretion in correlation with higher anti-HA2 and anti-M2e antibody responses.In the case of PRRSV, which was my main subject, I sought to optimize the IFNγ T cell responses by using DNA vaccines encoding antigens with conserved T-epitopes between strains, and targeted to APC. Indeed, whereas viral mutants escape neutralizing antibodies, it has been proposed that the IFNγ T cell responses are instrumental for cross-protection. I showed that the broadest T cell responses were induced by DNA vaccines combined to nanoparticles PLGA (NP) injected by the intradermal route, followed by electroporation (EP) compared with EP-only, intradermal route-only or transcutaneous dissolvable microneedles. This optimal immunogenicity was associated with a high transfection level of skin cells, an accumulation of inflammatory cells, and dendritic cells mobilisation. Next I used the EP+NP method to immunize pigs with plasmids encoding conserved PRRSV antigens targeted or not to APC via CD11c or XCR1. Pigs were immunized either with repeated injections of DNA alone or with a prime-boost DNA-MLV. The DNA-MLV regimen induced improved humoral and IFNγ T cell responses compared to DNA alone or MLV alone and the APC-targeting significantly increased the humoral response but not the IFNγ T cell response. Finally, I evaluated the DNA-MLV regimen efficacy, with an applied perspective, using naked DNA without NP and delivered by EP or by a convenient needle free injection technology (PJ). In these conditions, the DNA prime did not significantly increase the IFNγ T cell response induced by the MLV, but clearly increased the humoral response with a benefit of the APC-targeting. However, the immune potentiation induced by the DNA prime did not lead to an improved protection following a heterologous challenge. The heterologous protection was not correlated to the measured humoral and IFNγ T cell responses, and neutralizing antibodies were undetectable. Thus cross-protective effectors have not been sufficiently activated by our DNA-MLV strategy and the immune correlates of protection against heterologous PRRSV are still to be identified to develop cross-protective vaccines. Finally, this work shows that the effect of APC-targeting in pigs is influenced by delivery routes and methods and by vaccine regimen such as the prime-boost DNA-MLV

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Pliquet, Élodie. "Développement d’immunothérapies contre l’antigène de tumeur universel hTERT basées sur différentes stratégies vaccinales". Electronic Thesis or Diss., Paris 6, 2015. http://www.theses.fr/2015PA066747.

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Le cancer est l’une des principales causes de morbidité et de mortalité dans le monde. L’étude de ses mécanismes a mis en évidence des interactions particulières entre la tumeur et l’immunité adaptative. Les cellules cancéreuses suite à leur transformation maligne expriment des antigènes tumoraux reconnus par les LTs. Ce fondement constitue la base des immunothérapies ciblant les antigènes associés aux tumeurs (TAAs). Parmi les TAAs identifiés, la hTERT apparaît comme un antigène universel, par son implication dans le processus d’oncogenèse et sa surexpression dans 80 à 90% des cancers. Les réponses anti-hTERT trouvées chez des individus sains et des patients cancéreux, témoin d’un répertoire T spécifique préexistant et d’une cassure naturelle de la tolérance, ont orienté nos stratégies vaccinales sur ce TAA. Durant ce doctorat, des stratégies d’immunothérapies basées sur différents produits codant une forme inactive de la hTERT ont été développées. Le premier axe a constitué au développement de vaccins ADN thérapeutiques optimisés à la fois dans leur construction (délétions, réarrangements) et dans leur mode de délivrance. Une procédure d’électroporation a été mise au point afin de les délivrer efficacement dans le derme. Deuxièmement, dans l’optique d’augmenter l’immunogénicité de l’ADN par la réalisation de vaccinations hétérologues ou de créer un produit dérivé, un vecteur rougeole recombinant la hTERT a aussi été développé. Au cours de ce projet, l’immunogénicité et la cytotoxicité des réponses induites par la vaccination pour l’ensemble des constructions ont été démontrées in vivo dans des souris conventionnelles ou transgéniques HLA. De plus, un effet anti-tumoral a aussi été démontré pour le premier produit clinique d’Invectys
Cancer is one of the main causes of morbidity and mortality worldwide. Studies of the cancer process have highlighted specific interactions between tumor and adaptive immune system. Following their malignant transformation, cancer cells express tumor antigens which are recognized by T cells. This foundation is the basis of immunotherapy strategies targeting tumor associated-antigens (TAA). Among all identified TAAs, hTERT appears as a universal antigen which is also involved in oncogenesis and is overexpressed in 80 to 90% of cancer regardless of their histological origin. The hTERT specific immune response found in healthy volunteers and in cancer patients suggests a preexisting hTERT specific T repertoire and a natural break down of tolerance. In view of this, we focused our vaccine strategy on this TAA. During this PhD, immunotherapy strategies were developed based on different constructions which encoded modified inactive forms of hTERT. Firstly, we developed therapeutic DNA vaccines optimized both in nucleotide and amino acid sequence and for their delivery route. Nucleic or protein sequences were deleted or shuffled while retaining the predicted immunogenic parts of hTERT. A specific and safe electrogene transfer procedure has been established in order to deliver efficiently DNA vaccine in the dermis. Secondly with the aim of boosting vaccination to increase DNA immunogenicity, or to develop a derivative, a live-attenuated measles vaccine strains recombinant for hTERT was designed. For all constructs, we demonstrated the induction of specific hTERT immune responses which present cytolytic strength in vivo in wild-type or HLA transgenic mice. We also demonstrated the anti-tumor effect of Invectys’ first clinical product

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Nzonza, Angella. "Utilisation d'un rhabdovirus de poisson pour la mise en place d'une nouvelle plateforme vaccinale : exemple du virus West Nile (VWN)". Paris 7, 2013. http://www.theses.fr/2013PA077208.

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Le VWN est un arbovirus pouvant causer la maladie chez les mammiferes dont l'homme et le cheval. Notre projet a pour but de développer un vaccin vectorisé contre le VWN qui consiste à utiliser mi novirhabdovirus: le virus de la Septicémie Hémorragique Virale (VSHV). Il se réplique à des températures inférieures à 20°C et est naturellement inactivé à des températures au-delà. Un système de génétique inverse développé pour le VSHV, offre la possibilité d'ajouter des gènes dans le génome viral et de générer des virus recombinants rVSHV. Des virus recombinants exprimant d'une part le gène complet de la glycoprotéine d'enveloppe du VWN (Evwn) ou délété de sa région transmembranaire (rVSHV-EvwN et rVSHV-EvwnΔtm) et d'autre part des fragments codant certains domaines de la EvwN (soit le domaine III seul ou fusionné au domaine II) (rVSHV-DIIIvwN et rVSHV-DIIDIIIvwN, respectivement), insérés dans le génome du VSHV ont été générés. Le peptide signal ainsi que la région transmembranaire dérivés de la glycoprotéine G des Novirhabdovirus (PSG and TMG) ont été fusionnés aux antigènes du VWN aux extrémités amino et carboxy terminales respectivement pour optimiser leur adressage à la surface des virions. Nous avons démontré que la EvwN et le DIIIvwN pouvaient être exprimés à la surface du rVSHV lors de l'addition du PSG. Les constructions exprimant la EvwN fusionnée au PSG protégeaient la souris contre l'épreuve avec le VWN et spécifiquement rVHSV-PSgEvwn, qui induisait une réponse d'anticorps neutralisants corrélée avec la protection. Étonnamment, rVSHV exprimant le DIIIvwN ou le DIIDIIIvwN ne protégeaient pas de l'infection, bien qu'ils étaient fortement exprimés à la surface du virus
West Nile Virus (WNV) is an arbovirus that can cause disease in mammals including humans and horses. There is no specific treatment or vaccines for WNV in humans. Our study aims at developing a WNV vectored vaccine which consists in using a fish Novirhabdovirus vector: the Viral Hemorrhagic Septicemia virus (VHSV). VHSV replicates at temperatures lower than 20°C and is naturally inactivated at higher temperatures. A reverse genetics system has recently been developed for VHSV allowing the addition of genes in the viral genome and the generation of the respective recombinant viruses rVHSVs. We have generated rVHSV vectors bearing on the one hand the complete WNV envelope gene (EwNv) (rVHSV-EwNv and rVHSV-EwNΔtm) or deleted of his transmembrane domain and on the other hand, fragments encoding E subdomains (either domain III alone or fused to domain II) (rVHSV-DJIIwNv and rVHSV-DIIDIIIwNv, respectively) in the VHSV genome. With the objective to enhance the targeting of the EwNv protein or EwNv-derived domains to the surface of VHSV virions, Novirhabdovirus G-derived signal peptide and transmembrane domain (SPG and TMG) were fused to EwNv at its amino and carboxy termini, respectively. We demonstrated that both the EWNV and the DIIIwnv could be expressed at the viral surface of rVHSV upon addition of SPG. Constructs expressing EwNv fused to SPG protected mice against WNV lethal challenge and specifically rVHSV-SPGEwNv induced a neutralizing antibody response that correlated with protection. Surprisingly, rVHSV expressing EwNv-derived domain III or II and III were unable to protect mice against WNV challenge, although these domains were highly expressed at the viral surface

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Gravier, Rodolphe. "Biosécurité de la vaccination à ADN plasmidique chez le porc : prise en main du modèle (par étude de l’amélioration des voies vaccinales) et étude de la biodistribution et de la pharmacocinétique des plasmides". Rennes 1, 2007. http://www.theses.fr/2007REN1S054.

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Depuis quelques années, le laboratoire est impliqué dans l’étude de la vaccination ADN contre le virus de la pseudorage porcine (PRV) agent étiologique de la maladie d’Aujeszky. Des porcs sont injectés par voie intramusculaire profonde avec un mélange de trois ADN plasmidiques codant individuellement les glycoprotéines majeures gB, gC et gD du PRV (vaccin PRV-pcDNA3). Le but de ma thèse était dans un premier temps d’appréhender ce modèle de vaccination en regardant si la fusion de l’ubiquitine en N-terminal aux trois glycoprotéines du PRV (vaccin Ubi-PRV-pcDNA3) permettait d’améliorer la protection engendrée par rapport au vaccin ADN PRV-pcDNA3. Dans un deuxième temps nous avons étudié la distribution tissulaire et fait une étude pharmacocinétique préliminaire de chacun des trois plasmides contenus dans le vaccin PRV-pcDNA3
For several years, our laboratory has been involved in the study of DNA vaccination against pseudorabies virus (PRV), the etiological agent of Aujeszky’s disease in pigs. This technique consist in injecting pigs by deep intramuscular way with a mix of three plasmids encoding the three major PRV glycoproteins gB, gC and gD (PRV-pcDNA3 vaccine). My work was first to handle our vaccination model by looking if N-terminal fusion of ubiquitin to the three PRV glycoproteins (Ubi-PRV-pcDNA3 vaccine) could enhance protection compared to the DNA vaccine encoding the three native glycoproteins (PRV-pcDNA3). Secondly, we studied the tissue distribution and performed a preliminary pharmacokinetic study about each plasmid of the PRV-pcDNA3 vaccine

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Hervé, Maxime. "La glutathion S-transférase de 28kDa du schistosome : une enzime candidat vaccinal à l'interface des relations entre le parasite et le système immunitaire". Lille 2, 2002. http://www.theses.fr/2002LIL2MT11.

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Bruffaerts, Nicolas. "Preclinical studies on a new strategy combining the Bacillus of Calmette-Guérin with plasmid DNA-based subunit vaccines against tuberculosis". Doctoral thesis, Universite Libre de Bruxelles, 2015. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209082.

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La tuberculose est une maladie contagieuse causée par les bactéries appartenant au complexe Mycobacterium tuberculosis. On estime près de neuf millions de nouveaux cas et un million de décès chaque année dans le monde. De plus, approximativement un tiers de la population mondiale est infecté de manière latente, donc à risque de développer la maladie. Le seul vaccin préventif jusqu’à présent disponible est le Bacille de Calmette-Guérin (BCG). Cependant, son efficacité contre la forme pulmonaire de la maladie, contagieuse et plus fréquente chez l’adulte, est extrêmement variable. Le développement de nouveaux vaccins prophylactiques contre la tuberculose est basé sur une stratégie de remplacement ou d’amélioration de l’actuel vaccin BCG. De nombreux candidats vaccins sous-unitaires sont évalués dans un protocole de vaccination de rappel après le BCG. Ce dernier est en effet administré à plus de 80% des nouveau-nés et des nourrissons des populations à haut risque.

Le présent travail a eu pour but principal d’étudier une nouvelle approche de vaccination combinant le Bacille de Calmette-Guérin avec des vaccins sous-unitaires à ADN plasmidique dans différents modèles précliniques.

Plusieurs hypothèses tentent d’expliquer la faible efficacité du vaccin BCG, comme la faible induction de réponses immunitaires de type cellulaire T CD8+, le déclin de l’immunité protectrice induite au cours du temps, ou son répertoire antigénique limité. Les vaccins à ADN plasmidique induisant de telles réponses, le travail proposé a consisté au développement d’un nouveau protocole de vaccination basé sur la coadministration par la voie intradermique du vaccin BCG formulé avec un vaccin à ADN plasmidique codant pour un antigène mycobactérien. Nous avons observé dans plusieurs modèles murins (adulte et néonatal) une augmentation significative des réponses cellulaires de type CD4+ Th1 et CD8+, ainsi que de la réponse humorale spécifique. L’immunogénicité de cette approche a également été analysée dans un modèle animal de grande taille, à savoir le modèle porcin. Les résultats obtenus indiquent que les vaccins à ADN plasmidique sont capables d’augmenter les réponses spécifiques à l’antigène codé par le plasmide mais également celles spécifiques à d’autres antigènes exprimés par le vaccin BCG. Enfin, dans la deuxième partie du travail, nous avons développé des vaccins plasmidiques codant pour des combinaisons d’antigènes phase-spécifiques de M. tuberculosis et nous avons analysé leur immunogénicité en modèle murin.

En conclusion, nous avons montré que la stratégie de coadministration par la voie intradermique du vaccin BCG avec un vaccin à ADN plasmidique encodant des antigènes mycobactériens s’avère être un protocole de vaccination réaliste et efficace pour améliorer l’immunité induite par le vaccin BCG. Elle offre par ailleurs des perspectives pour être appliquée avec des plasmides codant pour des antigènes caractéristiques de la tuberculose latente, peu reconnus après vaccination BCG, pour protéger à la fois contre la tuberculose active d’une primo-infection et contre la réactivation d’une infection latente.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

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Adam, Lucille. "Dynamique de la réponse immunitaire précoce mise en place localement suite à l’injection d’un vaccin ADN associée à une électroporation chez le macaque cynomolgus". Thesis, Paris 11, 2014. http://www.theses.fr/2014PA114812/document.

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La compréhension des mécanismes immunologiques précoces mis en place suite à l’administration de vaccins est encore de nos jours largement méconnue. Pourtant de plus en plus d’études démontrent l’importance de ces mécanismes très précoces faisant intervenir les acteurs de l’immunité innée dans la génération d’une réponse spécifique efficace après vaccination. La peau est un organe intéressant pour l'administration de vaccins du fait de sa richesse en cellules présentatrices d'antigènes (APC), qui sont des cellules essentielles dans la mise en place de la réponse immunitaire. L'administration par voie intradermique du vaccin ADN de type auxoGTU induit des réponses immunitaires fortes et persistantes, en particulier en association avec une électroporation (EP) locale chez le macaque cynomolgus. Le but de ce travail de thèse fut de caractériser les réponses immunitaires locales précocement mises en place suite à l’administration par voie intradermique du vaccin ADN auxo-GTU en association avec une EP. Dans un premier temps, nous avons décrit les populations de cellules immunitaires présentes dans la peau normale chez le macaque cynomolgus.L'épiderme contient des cellules de Langerhans (LC) qui sont : CD1a+ CD1c- et des lymphocytes T caractérisés par l’expression du CD3. Le derme contient des cellules CD1a+CD1c-, qui présentent des similitudes avec les LC et correspondent donc probablement à des LC en migration à travers le derme. Il contient également des cellules dendritiques dermales (DDC) CD1a+CD1c+, des macrophages résidants CD163highCD11b+ et les lymphocytes T CD3+. Chez certains animaux, nous avons mis en évidence la présence de granulocytes CD66+ dans le derme sain. Les populations de cellules immunitaires identifiées chez le macaque sont similaires à celles identifiées chez l’homme malgré de légères différences phénotypiques. Cette caractérisation nous a ensuite permis d'étudier l’impact de la vaccination sur les populations immunitaires de la peau.Nous avons démontré que la vaccination induit le recrutement de granulocytes et de monocytes/macrophages inflammatoires dans l'épiderme et dans le derme, ainsi qu’un recrutement plus tardif de cellules dendritiques inflammatoires épithéliales (IDEC) dans l'épiderme. Dans l'épiderme, 24h après immunisation, nous avons observé une augmentation transitoire des LC accompagnée d’une surexpression de HLA-DR, de CD86 et de CD83, ce qui démontre leur maturation. Entre 24h et 72h, le nombre de LC diminue, ce qui suggère que les LC matures quittent l’épiderme pour migrer vers les nœuds lymphatiques. Ces événements cellulaires sont majoritairement dus à l’EP, indépendamment de la présence du vaccin à ADN. L’analyse du microenvironnement mis en place dans la peau suite à la vaccination révèle une libération de facteurs solubles pro-inflammatoires, comme MCP-1, IL-18 , IL-15, IL-8 et de facteurs solubles anti- inflammatoires comme IL-1RA et sCD40L dès 24h, dont certains sont considérablement augmentés par la présence de l’ADN vaccinal. Nos résultats suggèrent que l’EP, indépendamment de la présence de l'ADN, est suffisante pour induire la mobilisation des cellules et la maturation des DC au niveau du site de vaccination, ce qui montre un important rôle adjuvant de l’EP. Cependant, il semble que l'ADN soit nécessaire pour générer un microenvironnement favorable à l'activation optimale des APC. Ce travail fournit des éléments importants sur les mécanismes de l'inflammation locale et ouvre de nouvelles possibilités pour les stratégies vaccinales
Mechanisms involved in early vaccine response are poorly understood. However, more and more studies show the importance of innate immunity in the very early times following vaccine administration in the generation of an optimal specific immune response. Skin is an interesting target for vaccine delivery because of its richness in antigen presenting cells (APC) which are essential cells in immune responses. The intradermal delivery of auxoGTU DNA vaccine was shown to induce strong and persistent immune responses, especially in association with electroporation in cynomolgus macaque. The aim of this work was to characterize the early local immune responses followed intradermal auxoGTU DNA vaccination in association with EP in cynomolgus macaque. In a first step, we have described immune cell populations present in the normal skin in the cynomolgus macaques. The epidermis contains CD1a+CD1c- Langerhans cells (LCs), and CD3+ T cells. The dermis contains CD1a+CD1c- cells, which present similarities with LCs and probably correspond to LC in migration through dermis. It also contains CD1a+CD1c+ dermal dendritic cells (DDCs), CD163highCD11b+ resident macrophages, and CD3+ T cells. We found CD66+ polymorphonuclear cells in healthy dermis in some of the animals. Immune cell populations in the macaque are similar to those in humans despite moderate differences in phenotype. This characterization has allowed us to study the impact of vaccination on immune populations of the skin. We have demonstrated a recruitment of granulocytes and inflammatory monocytes/macrophages in epidermis and dermis, as well as a population of inflammatory dendritic epithelial cell (IDEC) in epidermis after vaccination. In epidermis, 24h after treatment, we have observed an initial increase of LC with an up-regulation of HLA-DR, CD86 and CD83, demonstrating their maturation. Between 24h and 72h, LC number decreased, suggesting that mature LC has leaved epidermis to migrate to skin draining lymph node. All these cellular events were almost due to EP process, independently of DNA vaccine presence. The skin microenvironment reveals a release of pro-inflammatory soluble factors, as MCP-1, IL-18, IL-15, IL-8 and anti-inflammatory mediators as IL-1RA and sCD40L by 24h, all considerably enhanced in the presence of DNA.Our results suggest that EP, independently of the presence of DNA, is sufficient to induce cells mobilization and DC maturation at the vaccinated site, suggesting an important adjuvant effect of EP. However, it seems that DNA is required to generate a favorable microenvironment essential for correct APC activation. This work provides important clues to local inflammation mechanisms and opens up new possibilities for vaccine strategies

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El, Mir Samir. "Lag-3 (cd223) : un ligand du CMH de classe II utilisé comme immuno-adjuvant de vaccination chez la souris". Paris 11, 2002. http://www.theses.fr/2002PA11T005.

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Le développement de nouveaux vaccins préventifs ou thérapeutiques requièrent très souvent la nécessité d'induire des réponses cellulaires CD8 et CD4 fortes et prolongées. Les sels d'aluminium utilisés actuellement comme adjuvants de vaccination chez l'homme ne sont utiles que pour induire des réponses humorales ou des réponses cellulaires de type Th2. L'induction de réponses CD4 Th1 et CD8 cytotoxiques chez l'homme à partir de fractions vaccinales sous-unitaires (ex : protéines recombinantes) requiert probablement, d'une part de nouveaux adjuvants (non-toxiques) capables de stimuler les cellules dendritiques interstitielles sous-cutanées vers une maturation de type DC1, d'autre part une formulation capable de vectoriser vers les cellules dendritiques et/ou protéger (effet dépôt) les protéines antigéniques. Mon travail de thèse a consisté à caractériser l'utilisation d'un ligand des molécules du CMH de classe II appelé LAG-3 (« Lymphocytes Activation Gene-3 », ou CD223) comme adjuvant permettant de rendre immunogénique des cellules tumorales ou des antigènes solubles ou particulaires de nature définie. Il a également porté sur la validation d'une nanoparticule cationique comme formulation permettant d'augmenter la bio-activité d'une protéine recombinante in vivo. Des cellules tumorales syngéniques murines transfectées par LAG-3 ou co-injectées avec une protéine de fusion recombinante soluble LAG-3Ig deviennent immunogéniques chez la souris avec induction d'une réponses CTL efficace permettant de rejeter la tumeur primitive ainsi que la greffe d'une nouvelle tumeur sauvage non immunogénique. Des réponses cellulaires CD8 et CD4 TH1 sont induites chez la souris immunisée avec un antigène de nature particulaire (HBsAg) ou soluble (OVA) lorsque l'adjuvant LAG-31g est additionné à l'antigène pour une injection sous-cutanée. Enfin, une nanoparticule de 60 nanomètres formée d'un cœur polysaccharidique cationique entouré d'une bi-couche lipidique a été validée chez la souris pour sa capacité à augmenter la bio-activité de l'IL-2 utilisée pour son action anti-tumorale passant par l'induction à de très faibles doses d'IL-2 de réponses CTL spécifiques d'antigènes de tumeur. Au total, ces résultats ont permis de poser les premières bases pour le développement à terme de lots cliniques de protéines recombinantes LAG-3 utilisées conjointement avec une protéine antigénique afin d'induire chez l'homme des réponses cellulaires CD8 et CD4 TH1 spécifiques de l'antigène. L'immunogénécité de ces assemblages LAG-3 / Ag pourrait être améliorée par leur formulation avec une nanoparticule cationique de type KY.

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Rochard, Alice. "Immunisation génétique par électroporation intramusculaire d'ADN plasmidique pour la production d'anticorps neutralisant la toxine botulique de type B". Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066711.

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Les neurotoxines botuliques (BoNTs), agents responsables du botulisme, sont les substances les plus toxiques actuellement connues. L'utilisation d'anticorps neutralisants pour l'immunothérapie passive est la seule solution retenue et accréditée par la FDA à l'heure actuelle. L'immunisation génétique par électroporation intramusculaire d'ADN s'est révélée être une alternative crédible à l'immunisation protéique pour produire des anticorps dirigés contre les sérotypes A et E des BoNTs avec un pouvoir neutralisant suffisamment élevé pour satisfaire aux exigences de la Pharmacopée Européenne. Cependant, les résultats se sont avérés décevants dans le cas du sérotype B, troisième type antigénique associé au botulisme humain. Comprendre la raison de la faible immunogénicité de BoNT/B en vaccination ADN et l'optimiser pour produire des anticorps neutralisants dirigés contre cette dernière ont constitué les deux objectifs de mon travail. En combinant différentes approches, nous avons montré que, contrairement à BoNT/A, la sécrétion de l'antigène BoNT/B est inhibée. Nous avons identifié le réticulum endoplasmique (RE) comme étant l'organite de rétention. Nous avons étudié un lien empirique entre la séquence protéique antigénique de BoNT/B et son accumulation intracellulaire et avons conclu que le mauvais repliement de la protéine antigénique était responsable de sa rétention dans le RE. Parmi les stratégies testées afin d'augmenter l'immunogénicité de BoNT/B en vaccination ADN, l'ajout de sites de N-glycosylation a permis de multiplier par 10 le pouvoir neutralisant des anticorps et l'utilisation des dérivés d'acides biliaires comme adjuvants de formulation est prometteuse
Botulinum neurotoxins (BoNT) have been characterized to be the most potent toxic substances identified so far. Passive immunization with antiBoNT antisera is the only treatment for botulism to have gained FDA approval. We have previously shown that genetic immunization by the non-viral intramuscular DNA electroporation technique is an effective alternative to recombinant proteins immunization to raise high titer neutralizing antibodies against BoNT serotype A and E. The neutralizing titers obtained were high enough to fit the European Pharmacopeia while it did not for type B, commonly linked to human disease as well. Finding out why BoNT/B immunogenicity is low after DNA vaccination and how we could improve it for generation of high-titer neutralizing antiBoNT/B antiserum were the two main goals of my work. Combining different approaches, we have first shown that BoNT/B antigen secretion was inhibited, while it did not for BoNT/A. We identified the endoplasmic reticulum to be the organelle of retention. Then, we studied an empirical link between BoNT/B antigenic protein sequence and intracellular accumulation. We concluded that protein misfolding could be the reason for BoNT/B retention in the endoplasmic reticulum. Among different strategies tested to improve BoNT/B immunogenicity in DNA vaccination, addition of N-glycosylation sites yielded 10 fold higher neutralizing antibodies titers. Finally, bile salts could be promising adjuvants for plasmid DNA vaccines

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Todorova, Biliana. "Imagerie in vivo de la réponse immune locale à la vaccination par voie intradermique à l’aide d’un ADN plasmidique associée à l’électroporation chez le macaque cynomolgus". Thesis, Paris 11, 2014. http://www.theses.fr/2014PA114837.

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L’électroporation (EP) in vivo est utilisée comme stratégie d’amélioration de la réponse immune induite par les vaccins ADN. Cependant son effet sur les acteurs du système immunitaire inné reste méconnu. Dans l’objectif de mettre en évidence le comportement cellulaire sur le site de la vaccination, nous avons développé des approches d’imagerie par fluorescence in vivo chez le macaque. Nos résultats montrent que l’EP locale, augmente non seulement la quantité et la distribution de l’antigène vaccinal, mais induit également la mobilisation et la migration des cellules de Langerhans. De plus, l’EP cause un recrutement de leucocytes dans la peau et le tissu sous-cutané et favorise la production de cytokines pro-inflammatoires dans la peau. Ces évènements précoces, qui résultent de l’utilisation de l’EP en tant que système de délivrance des vaccins ADN, mettent en évidence le potentiel de l’EP en tant qu’adjuvant vaccinal
In vivo electroporation (EP) is used as a strategy to improve the immune response induced by DNA vaccines. However, its local effect on the innate immune cells has not been fully described. We developed in vivo fluorescence imaging approaches to highlight the cell behavior in the site of vaccination in macaques. Our results show that the local EP not only increases the amount and the distribution of the vaccine antigen, but also induces the mobilization and migration of Langerhans cells. Furthermore, EP causes the recruitment of leukocytes into the skin and subcutaneous tissue and promotes the production of pro-inflammatory cytokines. These early events that result from the use of the EP as a delivery system for DNA vaccines, highlight its potential as a vaccine adjuvant

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Callendret, Benoît. "Conception et évaluation de différentes approches vaccinales contre le coronavirus associé au syndrome respiratoire aigu sévère". Paris 7, 2006. http://www.theses.fr/2006PA077222.

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Le coronavirus associé au syndrome respiratoire aigu sévère (SRAS-CoV) émergea à la fin de l'année 2002 et fut responsable d'une épidémie de pneumopathie atypique chez l'homme. Nous décrivons dans ce mémoire trois approches vaccinales contre le SRAS permettant l'induction d'anticorps neutralisants contre la glycoprotéine S, qui représentent les principaux effecteurs de la réponse immune protectrice. Nous avons montré que l'expression du gène S en cellules de mammifères nécessitait des vecteurs optimisés munis d'un intron et d'éléments de régulation post-transcriptionnelle comme WPRE ou CTE. Dans le modèle souris, seule l'association d'un intron et de WPRE permet d'augmenter l'immunogénicité d'un vaccin ADN contre le SRAS de telle sorte qu'il soit efficace à une dose faible d'ADN (2 μ g). Nous avons établi des lignées cellulaires sécrétant l'ectodomaine soluble de la protéine S (Ssol). L'immunogénicité du polypeptide Ssol purifié a été étudiée dans les modèles souris et hamster. Deux injections de Ssol adjuvé par de l'Alum permettent l'induction d'une réponse immune Th2 comprenant des titres élevés d'anticorps neutralisants, et protègent les animaux contre une infection d'épreuve par le SRAS-CoV. Nous avons également montré que l'utilisation d'adjuvants développés par GlaxoSmithkline Biologicals permettait une amélioration importante des réponses induites par la protéine Ssol et l'induction d'une réponse Thl. Parallèlement, nous avons montré chez le hamster qu'une seule injection de vecteurs lentiviraux permettant l'expression de la protéine S membranaire permet l'induction d'une réponse humorale neutralisante comparable à celle induite par deux injections de la protéine Ssol
Severe acute respiratory syndrome associated coronavirus (SARS-CoV) emerged in late 2002 and caused an epidemic of atypic pneumonia in humans. Here, we describe three vaccine candidates designed to induce neutralizing antibodies against the viral S glycoprotein, which are the main effectors of the protective immune response. We demonstrated that efficient expression of S gene in mammalian cell lines required the use of optimized vectors containing an intron and post-transcriptional regulatory elements such as WPRE and CTE. Upon immunization of mice with low doses of naked DNA, only intron and WPRE-containing vectors were able to provide protection against challenge with SARS-CoV. We also established stable cell lines constitutively secreting a soluble form of the S protein (Ssol). The immunogenicity of purified Ssol was studied in mouse and hamster models. Two injections of the Ssol polypeptide adjuvanted with Alum induced a strong and long-lasting Th2 immune response comprising high levels of SARS-CoV-neutralizing antibodies. Upon intranasal challenge with SARS-CoV, virus replication was strongly reduced in the lungs of immunized animals and hamsters were protected from the occurrence of lesions in the respiratory tract. Moreover, the use of two new adjuvants developed by GlaxoSmithKline Biologicals further increased the anti-S humoral response and the Thl component of the immune response. Concurrently, we developed HIV-based lentiviral vectors expressing the full-length S protein as an alternate SARS vaccine candidate. In the hamster model, a single injection of these vectors induced a neutralizing antibody response similar to that induced by two injections of Ssol

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Chrun, Tiphany. "Développement d’un vaccin à ADN optimisé contre le virus de la fièvre de la vallée du Rift chez le mouton". Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLA004/document.

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Transmis par les moustiques, le virus de la fièvre de la vallée du Rift (vFVR) est un virus zoonotique qui affecte principalement les ruminants en Afrique et conduit à des pertes économiques importantes. Il n’existe actuellement pas de traitements et les seuls vaccins disponibles sont à usage vétérinaire. Le développement de nouveaux vaccins plus sûrs contre le vFVR est une priorité de l’OMS en raison du risque d’émergence de cet arbovirus dans d’autres continents. Dans cette étude, nous avons développé une vaccination à ADN optimisée contre le vFVR qui consiste à administrer par voie cutanée un plasmide codant pour l’ectodomaine de la glycoprotéine de surface Gn du vFVR (eGn) en présence d’un plasmide adjuvant codant le GM-CSF et combinée avec une électroporation. De plus, nous avons également optimisé la vaccination à ADN en l’associant à la stratégie de ciblage des cellules dendritiques (DCs) via un plasmide qui code des fragments d’anticorps scFv fusionnés avec l’eGn dirigés contre les récepteurs DEC205 et CD11c exprimés à la surface des DCs. Les vaccins ont été testés chez le mouton, hôte naturel du virus et dans le modèle murin pour étudier les mécanismes de protection. Dans nos deux modèles d’études, l’immunisation par le plasmide codant l’eGn confère une meilleure protection après une épreuve virale ainsi qu’une forte production d’anticorps non neutralisants par rapport au ciblage des DCs. En revanche, le ciblage d’eGn vers des récepteurs de DCs protège partiellement contre une épreuve virale et induit une immunogénicité différente dans les deux espèces. Nous avons confirmé le rôle protecteur de ces anticorps anti-eGn par un transfert passif dans le modèle murin et le mécanisme d’action de ces anticorps protecteurs reste encore à être déterminé. Notre étude montre pour la première fois la protection par un vaccin à ADN contre le vFVR chez le mouton
The Rift valley fever virus (RVFV) is a mosquito-borne virus that mainly affect ruminants in Africa, resulting in economic burden. There is currently no treatment and only vaccine for veterinary use against the RVFV are available. The development of new and safer vaccine is urgently needed due to the risk of introduction of this arbovirus to other continents. In the present work, we developed an optimized DNA vaccination against RVFV using a plasmid encoding the ectodomain of surface glycoprotein Gn (eGn) of RVFV into the skin with plasmid adjuvant encoding GM-CSF and electroporation in sheep. We further optimized the DNA vaccination using dendritic cell targeting strategy with a plasmid encoding a single chain fragment variable (scFv) fused with eGn directed to two DC receptors, DEC205 and CD11c. The efficacy of the vaccines were tested in the sheep, the natural host and in the mouse model to investigate the mechanism of protection. In both models non-targeted eGn vaccine confer a better clinical protection and higher non-neutralizing antibody production than DC-targeted vaccine. However, in both models eGn targeting to DEC205 differentially affected the immune response and induced a partial protection after a challenge. We further demonstrated that non-neutralizing antibodies induced by native eGn protect mice by passive transfer. The mechanism mediated by these antibodies remains to be investigated. Overall, this work indicates the proof of concept that DNA vaccine can confer protection against the RVFV in the sheep

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Keita, Djénéba. "Utilisation de l’interférence ARN pour l’inactivation post-transcriptionnelle de gènes viraux et le contrôle de la réplication de deux virus animaux in vitro : Morbillivirus (ARN) et Peste Porcine Africaine (ADN)". Montpellier 2, 2008. http://www.theses.fr/2008MON20163.

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Ce travail avait pour objectif d'utiliser le mécanisme de l'interférence ARN pour le contrôle in vitro de la réplication de virus à ARN (Morbillivirus) et à ADN (Asfivirus). Par une approche bioinformatique et biologique par mesure de l'inhibition de la réplication de ces virus en culture cellulaire, des gènes cibles et des siARN actifs ont été mis en évidence. Le genre Morbillivirus inclus d'importants pathogènes de l'homme et de l'animal, comme le virus de la rougeole, de la peste des petits ruminants et celui de la peste bovine. La nucléoprotéine (N) joue un rôle central dans la transcription et la réplication des Morbillivirus, aussi, nous avons définis des siARN dirigés contre les séquences conservées déterminées par alignement multiple du gène N de ces virus. Dans le cas du virus à ADN étudié, le virus de la peste porcine africaine, l'objectif consistait à déterminer le rôle dans la réplication virale, de quatre gènes présents dans une région devant être délétée pour l'atténuation raisonnée du virus. Pour les pays confrontés à ces maladies virales extrêmement contagieuse, le développement de vaccins thérapeutiques reposant sur l'interférence ARN est un progrès majeur en santé animale, spécialement pour la peste porcine africaine contre laquelle il n'y a pas encore de vaccin et pouvant déboucher sur de nouvelles stratégies de lutte
This work aimed at using the mechanism of RNA interference for the in vitro control of the replication of RNA (Morbillivirus) and DNA viruses (Asfivirus). By bioinformatics and in vitro biological approaches measuring the inhibition of the replication of these viruses in cell culture, target genes and active siRNA were identified. Morbillivirus genus includes important pathogens of human and animals. They include measles virus, peste des petits ruminants virus and rinderpest virus. Nucleoprotein (N) plays an essential role in transcription and replication of Morbilliviruses, therefore we defined siRNA targeting the conserved sequences as defined by multiple alignment of the N gene of these viruses. In the case of the DNA virus studied, African swine fever virus, the objective was determining the role in the viral replication, of four genes present in an area having to be deleted for the carefully thought out attenuation of the virus. . For countries facing these extremely contagious viral diseases, the development of therapeutic vaccines based on siRNA interference is a major progress for animal health, especially for African swine fever against which there is not yet vaccine and having the potential to open the door on new control strategies

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Mirabelli, Carmen. "Exploring viral-host interactions during poliovirus infection : study of the role of the 3A cellular partners, ACBD3 and CREB3, in viral replication and cell signaling". Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCC128.

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Le poliovirus (PV) est un des prototypes de la grande famille des virus à brin positif d'ARN, les Picornaviridae, et il est l'agent étiologique de la poliomyélite paralytique. Bien qu'il soit le premier virus à avoir été cultivé, en 1949, et étudié in vitro, les connaissances de virologie fondamentale sur le PV sont encore lacunaires et incomplètes. Les campagnes de vaccinations mondiale, très efficaces, ont permis d'éradiquer la polio dans la plupart des grandes régions du monde, et la maladie ne reste aujourd'hui endémique que dans trois pays en Afrique et Asie. Si le PV n'est donc plus considéré comme une menace de santé publique en Europe et son étude n'est plus considérée comme prioritaire, il reste un modèle très captivant. En particulier, depuis l'introduction du vaccin oral antipoliomyélitique, la circulation et la dérive génétique de ces souches atténuées ont entrainé l'émergence de poliovirus dérivés du vaccin qui sont neuro-pathogènes. Ces souches sont le plus souvent recombinantes avec des virus co-circulants appartenant à la même famille que le PV, ce qui suggère que la recombinaison contribue au mécanisme d'émergence. Un des partenaires préférentiels de ces recombinaisons semble être le Coxsackievirus A17 (CV-A17). Mon projet de thèse vise à approfondir les connaissances actuelles du PV dans le cadre de son interaction avec la cellule hôte, avec notamment trois objectifs : Améliorer notre compréhension de la sélection des souches recombinantes dérivées du vaccin. Caractériser de nouveaux modules de signalisation cellulaire interagissant avec le PV. Identifier de nouvelles cibles pour le développement de thérapies antivirales qui seront nécessaires pendant la période de post-éradication. Une analyse du profil d'interaction virus-hôte de la protéine non-structurale 3A du PV nous a permis d'identifier deux nouveaux partenaires cellulaires :-ACBD3, une protéine qui joue un rôle dans le maintien de la structure de l'appareil de Golgi -CREB3, un facteur de transcription impliqué dans la réponse au stress du réticulum endoplasmique (RE) Pour ACBD3, nous avons montré que cette protéine module la réplication du PV et que son effet dépend de la nature de la protéine 3A. En effet, l'acquisition d'une 3A de CV-A17 confère au recombinant un avantage en termes de réplication, avantage dû à une moindre sensibilité à l'effet inhibiteur de ACBD3 sur la réplication (Téoulé F. Et al, Journal of virology 2013). Concernant le facteur de transcription CREB3, nous nous sommes intéressés à une de ses cibles : la protéine Herp, impliquée dans l'homéostasie calcique du RE, compromise pendant l'infection par le PV. Nous avons montré que le module de signalisation CREB3/Herp, activé par le PV aux temps précoces post-infection, limite l'augmentation du calcium cytosolique et l'induction de l'apoptose. Même si le rôle de l'interaction directe entre 3A et CREB3 n'a pas été élucidé, nous avons mis en évidence pour la première fois que le PV active une réponse cellulaire au stress RE. Toutefois, le PV modifie son aboutissement (notamment l'apoptose cellulaire), vraisemblablement pour permettre la réplication virale et éventuellement limiter l'étendue des lésions dans le système nerveux central. De plus, l'étude de CREB3 nous a amenés à identifier une cible cellulaire intéressante pour le développement de nouveaux antiviraux anti-PV : le RIP (module de protéolyse intra-membranaire régulé). Nous avons testé un médicament couramment utilisé comme inhibiteur de protéase dans le traitement de l'infection par le Virus de l'Immunodéficience Humaine de type 1 (VIH-1), le Nelfirlavir. Nous avons montré que celui-ci inhibe la réplication et la production de plusieurs PV d'origine vaccinale ou sauvage, et d'autres membres de la famille des Picornaviridae. Tous ces résultats confirment l'intérêt d'étudier les interactions virus-cellule infectée pour à la fois approfondir les connaissances de virologie fondamentale et pathogénèse du virus d'intérêt, étudier des mécanismes de sélection de souches mutées ou recombinées et éventuellement identifier des nouvelles stratégies antivirales
Poliovirus (PV) is a prototype member of the giant family of RNA+ single stranded virus, Picornaviridae, and it is the etiologic agent of poliomyelitis. Although PV has been studied sine 1949, the molecular virology of this virus has not been fully elucidated. The global vaccination campaign led to the complete eradication of PV in Europe and America and the virus remains endemic only in Nigeria, Afghanistan and Pakistan. Therefore, PV does not constitute a heath burden and a research priority anymore, but it still remains a captivating model of study. In addition, the externe plasticity of this virus together with sub-optimal vaccine coverage in some regions of the world permitted the emergence of new PV-derived pathogenic neurovirulent strains (VDPVs). In particular, these strains originate from the genetic drift of the attenuated oral poliovaccine (OPV) and episodes of genetic recombination with co-circulating enteroviruses (EV) of species C, in particular with the Coxsackievirus A17 (CV-A17). My thesis project focuses on the study of viral-host interaction during PV infection in order to : Propose mechanisms of emergence and selection of recombinant VDPVs Characterize new signaling modules interacting with PV Identify new cellular targets for the development of new PV antivirals, necessary during the post-eradication period The, analysis of the interaction maps of the non-structural protein 3A allowed us to identify new cellular partners: -ACBD3 Golgi-resident protein maintaining the structure of the Golgi organelle -CREB3 transcription factor involved in endoplasmic reticulum (ER) stress In this study, we showed that ACBD3 restricts viral replication and its effect depends on the nature of viral protein 3A. Indeed, the acquisition of a 3A derived from CV-A17 is beneficial for the replication of the recombinant virus, which is less sensitive to the inhibitory effect of ACBD3 (Téoulé F. Et al, Journal of virology 2013). CREB3 is an ER-stress induced transcription factor. One of its targets is the Herp protein, involved in ER calcium (Ca2±) homeostasis via the degradation of the ER-resident Ca2+ channels. During PV infection, a Ca2+ flux from the ER has been reported at late times post infection. In this study, we showed that the signaling module CREB3/Herp, activated at early times post infection, limits Ca2+ flux and PV-induced apoptosis. We provided evidences that PV activates an ER-stress response but modifies its outcome (cell apoptosis) to assist viral replication. Moreover, the anti-apoptotic pathway CREB3/Herp could limit viral-induced damages of the central nervous system. In addition, the study of CREB3 allowed us to identify a target for antiviral therapy: the regulated intramembrane proteolysis (RIP) pathway. We tested an inhibitor of the RIP, originally developed for HIV-1 as a protease inhibitor, Nelfinavir (NFV). Here, we reported the antiviral activity of NFV in vitro against PV and a panel of enteoviruses. Altogether, these results support the interest in studying viral-host interactions to deepen the fundamental knowledge on the molecular virology and pathogenesis of the virus, study new mechanisms of viral emergence and eventually identify new strategies to combat viral infections

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Rashid, Muhammad Imran. "RON4 et ROP18 deux protéines de rhoptries de Toxoplasma gondii candidats vaccins ? : Etude dans un modèle de toxoplasmose chronique chez la souris". Thesis, Tours, 2011. http://www.theses.fr/2011TOUR3804.

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Toxoplasma gondii, protozoaire intracellulaire obligatoire, est l’agent responsable de la toxoplasmose, infection qui revêt un caractère sévère au cours de toxoplasmoses cérébrales ou congénitales en médecines humaine et vétérinaire. Aucun vaccin n’est actuellement disponible ; le développement de stratégies vaccinales efficaces est donc d’actualité. Le potentiel vaccinant de deux protéines de rhoptries de T. gondii, RON4 et ROP18, protéines injectées dans la cellule au site de l’invasion lors de l’étape d’attachement à la cellule, a été évalué dans deux stratégies vaccinales contre la toxoplasmose chronique chez la souris: vaccination ADN par voie intramusculaire et vaccination par voie nasale avec des protéines recombinantes. L’immunisation avec des plasmides optimisés exprimant RON4, la partie N-terminale ou la partie C-terminale de RON4 co-administrés avec un plasmide exprimant l’adjuvant GM-CSF ou l’immunisation par voie nasale avec une protéine recombinante RON4 associée à la toxine cholérique, induit des réponses systémiques humorale et cellulaire (mixte Th1/Th2) mais ne confère pas de protection. Dans nos conditions expérimentales RON4 n’est pas un candidat vaccin potentiel. Des stratégies pour augmenter son immunogénicité par voie nasale et pour orienter la réponse cellulaire vers un profil Th1 pourraient cependant être envisagées. L’immunisation avec des plasmides bicistroniques exprimant à la fois ROP18 sous forme sécrétée et le GM-CSF ou ROP18 cytosolique et le GM-CSF, induit des réponses humorales et cellulaires (Th1) similaires et ne confère pas de protection significative. La co-administration d’un plasmide exprimant l’IL-12 n’augmente pas les réponses immunes avant infection mais a néanmoins contribué à augmenter la réponse cellulaire après infection. L’immunisation par voie nasale avec une protéine recombinante ROP18 associée à la toxine cholérique, induit une réponse systémique humorale (Th1/Th2) et confère une protection significative (réduction de la charge parasitaire de 50%). La co-administration de l’adjuvant poly I:C augmente la réponse cellulaire mais n’a pas d’effet sur la protection. Nos résultats suggèrent que ROP18 est un candidat vaccin potentiel, des stratégies pour améliorer son effet protecteur sont à envisager
Toxoplasma gondii, an obligate intracellular protozoan, is the etiologic agent of toxoplasmosis. This infection has severe consequences during cerebral or congenital toxoplasmosis both in human and veterinary medicines. No vaccine is currently available, so the design of efficient vaccine strategies is still a topical question. In this study, RON4 and ROP18, two rhoptry proteins of T. gondii which are discharged into the host cell at the invasion site, immediately following intimate contact with the host cell, were evaluated in two vaccine strategies against chronic infection in mice: DNA vaccination by the intramuscular route and recombinant protein vaccination by the nasal route. DNA immunization with optimized plasmids encoding full length RON4, or only the N-terminal, or the C-terminal part of RON4 plus a plasmid encoding the adjuvant GM-CSF or nasal immunization with a recombinant RON4 protein plus cholera toxin induced systemic humoral and cellular responses (mixed Th1/Th2) but failed to confer protection. Strategies intended to enhance the immunogenicity of RON4 by the nasal route and to enhance the Th1 immune response against RON4 could be more effective.DNA immunization with ROP18 expressed as a secreted or a cytosolic form by bicistronic vectors which encode both the antigen and the adjuvant GM-CSF induced similar humoral and cellular (Th1) responses but did not confer significant protection. Co-administration of a plasmid encoding the adjuvant IL-12 did not enhance the immune responses before challenge but was able to prime a cellular immune response that was boosted by the parasite infection. Nasal immunization with a recombinant ROP18 protein plus cholera toxin induced systemic humoral responses (mixed Th1/Th2) and conferred partial protection (50% brain cysts reduction). Co-administration of the adjuvant poly I:C enhanced the cellular response but did not potentiate the protection. Our data suggest that ROP18 is a potential vaccine candidate against toxoplasmosis. Strategies to improve the protective effect of ROP18 should be investigated

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Vidalin, Olivier. "Étude de l'immunogénicité des protéines structurales du virus de l'hépatite C par le biais de vaccinations génétiques". Lyon 1, 1999. http://www.theses.fr/1999LYO1T311.

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Guerenne, Laura. "Validation de modèles précliniques de transformation de Syndrome Myélodysplasique en Leucémie Aiguë Myéloide et application à l'étude de nouvelles thérapies". Paris 7, 2014. http://www.theses.fr/2014PA077116.

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Les syndromes myélodysplasiques (SMD) sont un groupe hétérogène de maladies clonales de la cellule souche hématopoïétique caractérisé par une dysplasie de la lignée myéloïde. Ces syndromes évoluent dans environ 40% des cas en leucémie aiguë myéloïde (LAM). L'étude des modèles animaux représente une opportunité de mieux comprendre la leucémogénèse myéloïde. C'est aussi le point de départ du développement de nouvelles thérapies afin de mieux traiter les patients atteints d'un SMD et d'une LAM post SMD. Nous avons réalisé un criblage génomique à grande échelle par puce à ADN afin d'obtenir le profil d'expression génique de deux modèles transgéniques de SMD et LAM post SMD. Ces profils ont été comparés avec le profil d'expression génique de patients afin de mettre en évidence leur pertinence pour l'étude des SMD et LAM post SMD de l'homme. Cette analyse a montré des similarités entre les profils d'expression des modèles et des patients et mis en avant des altérations de l'expression de gènes impliqués dans des fonctions cellulaires encore peu décrites comme impliquées dans le développement de la maladie. Nous avons également évalué l'efficacité d'un vaccin à ADN non spécifique, le pVAX14, et d'une molécule inhibitrice des protéines à domaine BH3 dont les protéines anti-apoptotiques de la famille BCL-2, l'ABT-737, dans différents modèles animaux. Dans un modèle murin transplantable de LAP et le modèle murin transgénique de SMD de haut risque, la vaccination par pVAX14 entraîne une prolongation de la survie ainsi qu'une activation de la réponse immunitaire humorale et cellulaire similaire à ce qui a déjà été observé dans le modèle murin LAP avec un vaccin spécifique exprimant un antigène du gènes de fusion PML-RARot. Ces résultats suggèrent une utilisation « générique » du pVAX14 dans d'autres types de cancers ou hémopathies malignes. L'ABT-737 quand à lui, améliore la survie dans le modèle murin transgénique de SMD de haut risque en ciblant les cellules initiatrices de la leucémie et les cellules progénitrices primitives, en régulant le cycle cellulaire, la différenciation et l'apoptose
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonai hematopoietic stem tell diseases characterized by dysplasia in the myeloid lineage. 40% of MDS patients evolve to an acute myeloid leukemia (AML). The study of animal models provides an opportunity to better understand the myeloid leukemogenesis. It is also the starting point for the development of nevi therapies to better treat patients with MDS and AML post a SMD. We conducted a large-scale genomic screening by DNA chip to obtain the gene expression profile of two transgenic mouse models of MDS and AML post MDS. These profiles were compared with the gene expression profile of patients in order to highlight their relevance to the study of MDS and AML post SMD. These analyses showed similarities between expression profiles of patient and mouse models and have shown alterations in expression of genes involved in cellular functions described as yet little involved in the development of the disease. We also assessed the efficacy of a DNA nonspecific vaccine, the pVAX14, and a molecule which inhibite proteins BH3 domain. ABT-737 has an effect on anti-apoptotic BCL-2 proteins family. In a mouse model of transplantable LAP and transgenic mouse model of high-risk MDS, vaccinatior with pVAX14 increase mice survival and activate the immune response as it was already shown in the LAP mouse model with a specific vaccine expressing PML-RAR alpha antigen. These results suggest a use "generic" pVAX14 to treat other types of cancer or haematological malignancies. ABT-737 improves the survival in the transgenic mouse model of high-risk MDS and target leukemia initiating cells and primitive progenitor cells, by regulating the tell cycle, differentiation, and apoptosis

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27

Vignes, Caroline. "Étude du rôle de la réponse T dans le rejet et la tolérance d'allogreffe : ciblage de la réponse T par vaccination anti-TCR avec de l'ADN nu". Lyon 1, 1999. http://www.theses.fr/1999LYO1T131.

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Jayaraj, Ramamoorthi, i Jayaraj@menzies edu au. "Expression of stage-specific Fasciola proteases and their evaluation in vaccination trials". RMIT University. Applied Science, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20081029.100156.

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The liver flukes Fasciola hepatica and F. gigantica cause infectious disease in ruminants and humans. The geographical range of these two parasite species (temperate and tropical respectively) ensures that infection can occur worldwide. Although anthelmintic treatment is effective against disease, emerging drug resistant strains leads to the development of a vaccine. However, despite several decades of research, there is no commercial vaccine available. The main challenge at present is to produce recombinant proteins in an immunologically active form using recombinant DNA technology. This is an essential step in Fasciola vaccine production. Cysteine proteases are probably the most important facilitators of virulence in flukes and are produced by all stages of the fluke life-cycle. Two classes of cysteine protease are found in the excretory and secretory material of liver flukes- these are cathepsin L and cathepsin B. As such, the major aims of this thesis were to investigate the expression and purification of Fasciola recombinant cysteine proteins, and characterisation by SDS-PAGE and immunoblotting using monoclonal and polyclonal antibodies. These studies demonstrate the production of functionally active cathepsin proteins in S. cerevisiae BJ3505 cells which will lead to vaccine candidate analysis. The second aim of this thesis was to determine the protective efficacy of stage specific target antigens against experimental infection. In addressing this issue, the protective efficacy of single and multivalent recombinant protein vaccinations of adult stage F. hepatica cathepsin L5, immature F. gigantica cathepsin L1g and juvenile F. hepatica cathepsin B were analysed in Sprague Dawley rats against F. hepatica infection. This study demonstrates that juvenile fluke target antigen-cathepsin B induces better immune protection than adult fluke antigen-cathepsin L5. Cocktails of juvenile and adult stage fluke recombinant proteins (cathepsin B and L5) elicited the highest protective immunity against experimental infection and this combination showed not only reduction in fluke recovery and size of flukes, but also marked diminution in the intensity of liver lesions in vaccinated rats. In order to assess the immunogenic property of an early infective stage fluke secreting cysteine protease as a vaccine candidate, DNA vaccination vectors encoding cathepsin B were analysed in BALB/c mice. In this study, the ability of four DNA vaccination strategies such as secretory, chemokine-activating, lymph node targeting vectors encoding cathepsin B were assessed by antibody titre, antibody avidity, western blotting and ELIPSOT assay. The results have further validated the immunoprophylactic potential of a cathepsin B vaccine against F. hepatica. In this study, we have expressed and attained high yields of F. gigantica cathepsin L1g from E. coli BL21, and compared this to a yeast-expressed system. This protease was over-expressed and formed insoluble inclusion bodies that were subsequently solubilised with urea or guanidine hydrochloride. In order to purify the urea-solubilised protein, step-wise urea gradient chromatography was used. For refolding of solubilised protein, a dilution and dialysis procedure was utilised. Proteolytic activity was confirmed by gelatin SDS-PAGE analysis. In conclusion, the determination of the immune potential of recombinant stage specific antigens allows the development of effective vaccines against Fasciola infection.

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Baynam, Gareth. "Genetic influences on vaccine response in children". University of Western Australia. School of Paediatrics and Child Health, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0259.

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Vaccination is one of the most efficacious public health interventions1 and has been increasingly used to combat non-infectious diseases. Mechanisms underlying vaccine responses overlap with those regulating immune responses in health and disease. Therefore, an understanding of mechanisms underpinning these responses will have broad implications. Variation in immune response genes contributes to impaired vaccine responses2-4. Understanding the contribution of genetic variants to vaccine responses is likely to be particularly important in early life given the generalized functional immaturity of the immune system in infants and the highly variable kinetics of its maturation over the first few years of life5-7. However, studies of genetic influences on early childhood vaccine responses are scarce. Since a number of genes from several pathways are likely to be important, a targeted approach is necessary. This thesis explored the effects and interactions of genes associated with atopy, as atopy, or the genetic risk for it, has been associated with modulation of early childhood vaccine responses. This thesis aimed to: 1) investigate genetic variants associated with atopy on early childhood vaccine responses; 2) examine interactions between these genetic variants and non-genetic factors; 3) approach developmental genetic influences on genetic effects and their interactions; and 4) extend findings on vaccine responses to other immunological phenotypes and disease outcomes.

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Saxena, Manvendra, i s3031657@student rmit edu au. "Utilising salmonella to deliver heterologous vaccine antigen". RMIT University. Applied Sciences, 2007. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080522.095907.

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Live attenuated Salmonella vectors provide a unique alternative in terms of antigen presentation by acting as a vector for heterologous antigens. The efficiency of any live bacterial vector rests with its ability to present sufficient foreign antigen to the human or animal immune system to initiate the desirable protective immune response. Salmonella vectors encoding heterologous protective antigens can elicit the relevant immune responses, be it humoral, mucosal or cell-mediated. STM-1 is a Salmonella mutant developed by RMIT, harbours a mutation in the aroA gene that renders it attenuated, and is a well characterised vaccine strain currently in use to protect livestock against Salmonella infection. In previous work in this laboratory, STM1 was shown to be capable of eliciting immune responses in mice to plasmid-borne antigens. In this study STM-1 was analysed for its ability to vector the model antigen chicken ovalbumin and test antigen C. jejuni major outer membrane protein using in vivo inducible promoters such as pagC and nirB from the plasmid location. The determination of the architecture around the lesion in STM-1 also allowed the development of constructs expressing heterologous antigen from the chromosome. The induction of immune responses, both humoral and cell mediated, was analysed. Another issue addressed in this study was effect of pre-existing immune responses in the animal host against the vector or related strains and the effects on generation of immune responses against the subsequently vectored antigen. Humoral and cellular immune responses to vectored ovalbumin and C. jejuni Momp antigens were observed following vaccination with STM-1, when antigens were expressed from either the plasmid or chromosomal location. Up-regulation of immune responses, both humoral and cell mediated, was observed against the vectored antigens in animals which were pre-exposed to either the bacterial vector or related strains. These results indicate that STM-1 has the potential to be used as a vector to deliver heterologous vaccine antigens from a single copy gene in the field. Lastly, the results from this study indicate that pre-existing immune responses against the bacterial vector or a related strain do in fact enhance both humoral and T cell responses against the heterologous antigen.

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Taylor, Kim, i kim taylor@y7mail com. "Evaluation of the vaccine potential of malarial TCTP". RMIT University. Applied Sciences, 2009. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20091020.112823.

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Malaria is a widespread parasitic disease, causing 300-500 million infections per year and resulting in over 1 million deaths. There is widespread resistance of the parasite to most of the antimalarial treatments available, indicating the need for a vaccine (http://www.rbm.who.int/wmr2005/). The translationally controlled tumour protein (TCTP) family are highly conserved eukaryotic proteins that have been assigned a variety of functions. While most studies have focused on the intracellular functions of TCTP, human, malarial and other parasitic TCTPs have also been reported to have extracellular functions in the induction of histamine release from immune cells (e.g. MacDonald et al., 2001; Rao et al., 2002). Malarial TCTP has been detected in the sera of malaria-infected individuals (MacDonald et al., 2001) and is also known to bind to the antimalarial drug artemisinin (Bhisutthibhan et al., 1998). In this study, TCTP was investigated as a malarial vaccine candidate due to a previously observed protective effect in mice infected with Plasmodium yoelii YM. In that study, PfTCTP immunisation conferred a significant delay in disease progression, as judged by reduced parasitemia and prolonged survival (Taylor, 2002). It was thought that the protective effect might have been due to the inhibition of the extracellular actions of malarial TCTP by the acquired host immune response. P. falciparum and P. yoelii TCTP were initially expressed in S. cerevisiae, as in the previous study. The recombinant proteins were used to vaccinate mice, which were then challenged with two strains of P. yoelii. No protective effect was observed for either vaccine, and so the previous results using PfTCTP could not be confirmed. The TCTP of P. yoelii and P. berghei were then expressed in E. coli, which increased yield and decreased proteolysis. The recombinant proteins were used as vaccines in mice challenged with P. yoelii YM, P. c. chabaudi AS, or P. berghei ANKA. A significant delay in disease progression was observed in PyTCTP-immunised mice challenged with the non-lethal P.c. chabaudi, as determined by a significantly reduced parasitemia at each day post-infection leading up to a delayed peak parasitemia. A significant reduction in parasitemia was also observed in the early stages of P. yoelii YM infection in PyTCTP-immunised mice. P. berghei ANKA was used to challenge C57BL/6 mice to determine whether PbTCTP immunisation could protect mice from cerebral malaria development, no protective effect was observed. P. berghei ANKA was also used as a second lethal malaria challenge model in BALB/c mice, no significant differences in disease progression were observed in immunised mice. To further assess the functions of malarial TCTP, several attempts were made to create a TCTP-knockout strain of P. berghei ANKA. A TCTP-knockout malaria strain could be assessed for alterations in morphology, infectivity and artemisinin sensitivity compared with wild-type parasites. Initial genotype analysis of parasites resulting from several transfection experiments indicated that TCTP disruption had been successful, however TCTP-disrupted parasites were strongly selected against, and stable knockout strains could not be obtained. This indicates that TCTP performs an important role within the malaria parasite.

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32

au, w. ditcham@murdoch edu, i William Ditcham. "The development of recombinant vaccines against Jembrana disease". Murdoch University, 2007. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20071119.94111.

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Jembrana disease virus (JDV) is a lentivirus causing an acute infection with a 17% case fatality rate in Bali cattle in Indonesia. Control of the disease is currently achieved by identification of infected areas and restriction of cattle movement. A detergent-inactivated whole virus tissue-derived vaccine is sometimes employed in affected areas. This thesis reports initial attempts to produce genetically engineered vaccines to replace the inactivated tissue-derived vaccine, which as it is made from homogenised spleen of infected animals, is expensive to produce and could contain adventitious agents present in the donor animals. 4 potential DNA vaccine constructs were created containing the JDV genes coding for the Tat, capsid (CA), transmembrane (TM) and surface unit (SU) proteins in a commercially available vaccine plasmid. These were assessed for functionality in a range of in vitro and in vivo assays. All proteins were expressed in vitro and administration of 2 of the constructs by a commercial gene gun into the epidermis of mice resulted in antibody production to the appropriate protein. Due to the difficulties of licensing such a DNA vaccine in Indonesia, these vaccines were not progressed further. A mathematical model was developed to describe the progression of the acute phase of Jembrana disease following experimental infection with JDV. The model divided the disease into 6 phases based on the rates of viral replication and clearance calculated from data on sequential plasma viral RNA load detected by quantitative reverse-transcription polymerase chain reaction. This allowed statistical comparison of each phase of the disease and comparison of the severity of the disease process in groups of animals. The use of the model overcame the difficulty of comparing the disease in different animals as a consequence of the animal-to-animal variation in the disease process. The mathematical model was used to identify differences in the pathogenicity of 2 strains of JDV. One strain, JDVTAB caused a more rapid onset of disease in non-vaccinated controls, a significantly higher virus load at the onset of the febrile period and a higher peak viraemia than in animals infected with JDVPUL. This provided the first evidence of variation in pathogenicity of JDV strains. The measurement of virus load also demonstrated that some JDV infected animals developed a clinical disease that was not typical of that which had been reported previously. When infected with less than 1,000 infectious virus particles, up to 20% of infected animals failed to develop a febrile response. Infection of these animals was confirmed, however, by the detection of a high titre of circulating virus particles in plasma. These atypical infections had not been reported previously. Application of the mathematical model describing the progression of the disease in individual animals was used to examine the effect of vaccination with the inactivated tissue-derived vaccine on the progression of the disease. Several effects were noted in vaccinated animals that were subsequently infected with JDV: a reduction in the duration of the febrile response, a reduction in the severity of the febrile response in the early phases of the acute disease, and a reduction in virus load in the early and later phases of the disease process. The effect of vaccination with recombinant Tat, matrix (MA) and CA protein vaccines expressed in a bacterial expression system on subsequent JDV infection was also examined. A vaccine incorporating recombinant Tat and CA vaccine emulsified with Freunds incomplete adjuvant decreased the febrile response particularly in the later stages of the acute disease process, decreased the severity of the leucopenia in the later phases of the acute disease, and decreased the virus load in some but not all phases of the acute disease process. Vaccines administered with Freunds incomplete adjuvant were more efficacious than vaccines administered with QuilA, the latter actually exacerbating the disease process in vaccinated animals.

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Saade, Fadi. "Évaluation de nouvelles combinaisons immunothérapeutiques à base du vaccin à ADN nu pour le traitement des hépatites B chroniques". Lyon 1, 2008. http://www.theses.fr/2008LYO10047.

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Malgré la disponibilité d’un vaccin prophylactique efficace, l’infection par le virus de l’hépatite B (HBV) demeure un problème majeur de santé publique ainsi qu’un défi thérapeutique. La thérapie vaccinale à base d’ADN nu est prometteuse pour le traitement des hépatites B chroniques. Toutefois, il serait nécessaire d’améliorer son efficacité thérapeutique pour permettre son utilisation ultérieure en clinique. L’objectif de ce travail était d’utiliser le modèle du HBV de canard (DHBV), apparenté au virus humain, pour évaluer les bénéfices de la co-administration génétique de cytokines (IL-2, IFN-γ) avec des plasmides exprimant des protéines structurales de DHBV, sur l’augmentation de l’efficacité du vaccin. Dans une 1ère étude, nous avons évalué, chez des animaux naïfs, les bénéfices de la co-administration de plasmides exprimant l’IL-2 ou l’IFN-γ (pCI-IL2, pCI-IFNγ respectivement) avec un vaccin à ADN exprimant la grande protéine d’enveloppe du DHBV (pCI-preS/S), sur la réponse humorale neutralisante. La co-immunisation avec pCI-IL2 ou pCI-INFγ augmente les titres et le potentiel neutralisant des anticorps anti-preS, comparée à l’immunisation avec pCI-preS/S seul. Dans une 2ème étude, nous avons testé chez des canards chroniquement infectés l’efficacité de la thérapie associant la co-administration de pCI-IL2 ou pCI-IFNγ avec un vaccin à ADN exprimant les protéines preS/S et core. Nous avons démontré que la co-immunisation avec pCI-IFNγ entraîne une élimination plus marquée de l’ADN viral sérique, associée à une séroconversion avec des titres anti-preS significativement plus élevés (P< 0. 05), comparés aux autres groupes traités. De plus, l’ADN viral intrahépatique, ADNccc y compris, étaient indétectables par des méthodes conventionnelles chez 25% et 57% des animaux co-immunisés avec pCI-IL2 et pCI-IFNγ respectivement. D’une manière intéressante, l’inoculation à des canetons nouveau-nés, des extraits hépatiques provenant de 7 animaux qui avaient apparemment résolu l’infection, gardant des traces d’ADNccc détectables uniquement par PCR en temps réel, a montré l’absence d’infectivité pour 4 foies, alors que les extraits hépatiques des 3 autres animaux ont induit une infection associée à une forte réplication virale chez des canetons nouveau-nés. L’infectivité des extraits hépatiques était associée pour 2 animaux à une expression hépatique évidente des antigènes viraux. Dans une 3ème étude, nous avons réalisé et validé différentes constructions d’adénovirus aviaires « CELO » (Chicken Embryo Lethal Orphin) recombinants exprimant la protéine preS/S et l’IFNγ respectivement. Nous avons déterminé la voie d’administration optimale de ces vecteurs qui permet l’induction de la réponse humorale la plus puissante. Cette validation a permis d’initier un protocole thérapeutique « prime-boost » associant une primo-immunisation « prime » par un plasmide codant la grande protéine d’enveloppe du DHBV suivie d’injection de rappel hétérologue « boost » par des adénovirus CELO recombinants exprimant l’enveloppe du DHBV seule ou en combinaison avec un adénoCELO exprimant l’IFN-γ. En conclusion, notre étude a démontré l’effet bénéfique de l’adjonction de l’IFNγ avec un vaccin à ADN nu, permettant d’améliorer la rupture de l’immunotolérance et la clairance virale chez un grand nombre d’animaux. Ces résultats sont particulièrement intéressants pour le développement de la thérapie vaccinale à base d’ADN nu chez des porteurs chroniques de HBV
In spite of the availability of an efficient prophylactic vaccine, hepatitis B virus (HBV) infection remains a major public health problem and a therapeutic challenge. DNA-based vaccine is a promising strategy for chronic HBV infections treatment, although it is crucial to improve its efficacy. The aim of this work was to assess the therapeutic benefits of co-administration of cytokine genes (IL-2, IFN-γ) with plasmids expressing DHBV proteins, using the duck HBV (DHBV) infection model, closely related to the human virus. In a 1st study, we explored first in naïve ducks, the impact co-delivery of IFN-γ or IL-2 encoding plasmids (pCI-IFNγ, pCI-IL2 respectively) on humoral neutralizing response induced by DNA-based vaccine encoding DHBV preS/S large envelope protein (pCI-preS/S). Co-delivery of either pCI-IL2 or pCI-IFNγ considerably increased the magnitude and the neutralizing efficacy of anti-preS humoral response, as compared to duck group immunized with pCI-preS/S alone. In a 2nd study, therapeutic efficacy was tested in chronic-DHBV carrier ducks receiving envelope and capsid expressing plasmids (pCI-preS/S, pCI-C) alone or in co-immunization with pCI-IFN or pCI-IL2 plasmids. Co-delivery of pCI-IFNγ led to a significantly lower mean viremia, associated with seroconversion to higher anti-preS titers (P< 0. 05) compared to other groups. Moreover, liver DHBV DNA, including cccDNA, was undetectable by conventional methods for 25% and 57% of animals co-immunized with IL-2 and IFN-γ, respectively. Inoculation of liver homogenates from 7 resolved animals, presenting cccDNA detectable by real-time PCR only, showed absence of infectivity for 4, however 3 induced high titer viremia in neonatal ducklings, associated with evidence of intrahepatic preS expression for two animals. In a 3rd study, we realized and validated different constructs of recombinant avian adenovirus "CELO" (Chicken Embryo Lethal Orphin) expressing the preS/S and IFN-γ proteins respectively. We determined the optimal administration route of these vectors which allowed induction of highest anti-preS antibody response. This optimization allowed us to initiate a prime-boost therapeutic protocol, associating a prime with naked DNA vaccine targeting DHBV large envelope protein, followed by boosts with recombinant adenovirus CELO expressing this protein alone or in combination with IFN-γ. In conclusion, co-delivery of IFN-γ plasmid enhanced therapeutic efficacy of DHBV DNA vaccine in terms of break of humoral immune tolerance and viral clearance. These results are of particular value for the development of DNA vaccine-based immunotherapy for HBV-chronic carriers

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Xue, Lumin, i Lumin Xue@csl com au. "Immunological studies of cold-adapted influenza vaccine viruses in mice". RMIT University. Applied Sciences, 2009. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20091027.101804.

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Cold-adapted (ca) live attenuated influenza vaccines (LAIVs) have been introduced as alternatives to existing inactivated influenza vaccines. The influenza A components of the FDA-approved ca LAIVs (Flumist®; Medimmune) have common internal genes derived from the donor strain A/Ann Arbor/6/60 ca and surface genes derived from current wild-type (wt) epidemic strains. The aim of this thesis was to investigate determinants of immunogenicity for reassortants of A/Ann Arbor/6/60 ca, using a range of immunological assays, including recently developed MHC tetramer techniques. From the study, the extent of viral replication in the respiratory tract of mice, the primary site of inoculation, was a key factor in determining ca vaccine immunogenicity. Replication was shown to be influenced by both viral surface Ags and the host MHC. The H3 ca reassortants CR6, CR18, CR29 and CR6-35* exhibited greater replication efficiency (as determined by their PFU:HAU ratios) than the H1 ca reassortants CR35 and CR6-35. The H3 ca reassortant CR6 caused a 3.79% loss in body weight but no losses were observed for the H1 ca reassortant CR35 and the ca H2N2 donor strain A/Ann Arbor/6/60 ca. Higher HI responses were detected after 3 weeks in groups infected with the H3 ca reassortant CR6 (GMT 80) than with the H1 reassortant CR35 (GMT 10) and the H2 ca donor strain A/Ann Arbor/6/60 ca (GMT 13). Recently developed techniques were used to evaluate specific T-cell response to ca LAIVs. Fluorescent-labelled tetramer is the key reagent for use in tetramer-based flow cytometry assays. The NP366-374 peptide of influenza A viruses comprises an immunodominant epitope that is highly conserved between subtypes. Tetramers developed for A/PR/8/34 (H1N1) were able to detect NP-specific cytotoxic T lymphocytes (CTLs) induced by A/Ann Arbor /6/60 ca (H2N2). An attempt to prepare the A/Ann Arbor/6/60 ca-specific-NP-tetramer is described. H-2Db monomers were successfully refolded with the peptide, but only 20% were able to form tetramers through biotin-streptavidin linkage, resulting in a poor capacity to stain. By contrast, an IFN-γ ICC assay developed in parallel demonstrated that peptide NP366-374 was able to restimulate A/Ann Arbor/6/60 NP ca-specific CTLs and secrete IFN-γ when tested in vitro. Specific-B and T cell responses induced in the lungs in response to infection by ca reassortants exhibited great variability that was determined by the growth characteristics of different viruses. Type I (CTL) responses were induced by low yielding ca reassortants, such as CR35 (H1N1). Viruses with enhanced growth characteristics, such as CR6 (H3N2), produced higher Type II (HA-specific Ab) responses. In addition, host factors, such as MHC type, were found to play an important role in responses to the same viruses. Susceptible mouse strains, such as C57BL/6, showed higher CTL but lower serum Ab responses than more resistant strains, such as BALB/c. Throughout this PhD project, a fine balance between the humoral and CMI, local and systemic immune responses induced by ca LAIVs was demonstrated. The need to assess local immune responses, in addition to serum antibody levels, for the evaluation of vaccine efficacy was an important conclusion of the thesis.

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Wilson, Sarah, i n/a. "Vaccine peptide delivery by virus particles". University of Otago. Department of Microbiology & Immunology, 2007. http://adt.otago.ac.nz./public/adt-NZDU20080131.161222.

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Vaccination with immunogenic peptides offers a safe and specific way of inducing protection against pathogens, however as of yet there are no peptide-based vaccines available. The limitations on the therapeutic use of peptides are due to their poor immunogenicity and short life span in vivo. Peptide delivery systems act to circumvent these issues. The aims of this research were to investigate the ability of virus-like particles (VLP) from Rabbit haemmorhagic disease virus (RHDV) to deliver immunogenic peptides, to characterize the immune response to these particles, and to investigate whether baculovirus could also act as a delivery system. The vaccine peptides HAT (representing a T helper cell epitope) and HAB (representing the major B cell epitope) derived from the haemagglutinin antigen of influenza virus A/PR/8/34 were used as a model to investigate the ability of these virus particles to act as delivery vehicles to the immune system. A scheme for the production and purification of RHDV VLP was established. Expression of the capsid protein from RHDV in a serum-free recombinant baculovirus system using suspension cultures of up to 200 ml, and separation by isopycnic centrifugation on cesium chloride gradients led to high yields of purified RHDV VLP. Up to 20 mg of pure VLP could be obtained from an 800 ml culture of insect cells infected with recombinant baculovirus. In vitro testing revealed that RHDV VLP carrying the peptide HAT as a genetic fusion were processed by dendritic cells (DC), and that this peptide could be presented to induce activation of T cells. However, the purified RHDV VLP alone were not able to induce significant upregulation of cell activation markers CD40, CD86, and CD80. A preliminary in vivo study revealed that when RHDV VLP carrying the HAT peptide were delivered by an intraperitoneal injection in the absence of adjuvant, the immune response to the peptide was weak, therefore the route of delivery and the use of immune adjuvants with the VLP were optimised. Five different routes of delivery and two different immune adjuvants were compared. VLP were delivered through subcutaneous, intraperitoneal, transcutaneous, intramuscular and intranasal routes. Delivery of the VLP through each of these routes resulted in potent serum antibody responses. However, the strongest antibody responses were elicited when the VLP were delivered through the intraperitoneal or intranasal routes. Of these two routes, intranasal delivery gave the best mucosal responses at the lung surface, and was therefore chosen as the route of delivery for subsequent trials. CpG DNA and the wild-type baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) were tested as adjuvants for the RHDV VLP. These two adjuvants gave similar results, both acting to enhance a T[H]1 type response against the VLP, characterized by significantly increased levels of serum IgG2a and enhanced IFN-γ production. Two approaches were then tested: using the RHDV VLP as a peptide carrier with a CpG adjuvant, and using baculovirus particles directly as self-adjuvanting carriers for vaccine peptides. HAT and HAB peptides were chemically coupled to RHDV VLP. Mice that were vaccinated with these VLP mixed with a CpG adjuvant were able to raise low levels of specific antibody in the serum against influenza, and specific IgA against influenza was detected in the lung. These results indicated that, though the immune responses raised were modest, the RHDV VLP was able to deliver the vaccine peptides to the immune system. HAT and HAB peptides were chemically coupled to baculovirus particles. When mice were immunized with the baculovirus carrying the vaccine peptides, they raised significant levels of IgG1 (p<0.001) and IgG2a (p<0.05) against influenza in the serum, when compared to peptide delivered alone. A significant level of influenza-specific IgA was also detected in the lung at 10 ng/ml in the mice that received the baculovirus coupled with peptide. Analysis of splenocyte cytokines showed that these mice also responded to restimulation with IFN-γ production at around 100 pg/ml. This research revealed that RHDV VLP are able to act as carriers for vaccine peptides, however there are some limitations to their use with the HAT and HAB model peptides. It also showed that baculovirus can be rapidly modified to carry vaccine peptides by chemical conjugation, and that these peptides can be delivered to induce specific systemic and mucosal immunity, raising both B cell and cell mediated responses. Both virus particles have potential as components for new strategies for vaccination.

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Macedo, Gonzales Rodney. "Development of therapeutic vaccine strategies and pre-clinical animal tumor models for head and neck cancers". Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066269/document.

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Les cancers des voies aéro-digestives supérieures, liés à la consommation d'alcool et de tabac mais également à l'HPV-16, ont un pronostic médiocre malgré les traitements actuels. Le développement de nouvelles stratégies innovantes dans des modèles précliniques adaptés est ainsi nécessaire. Nous avons préalablement développé une stratégie vaccinale ADN permettant l'auto-assemblage in vivo de pseudo-particules virales non infectieuses exprimant l'oncoprotéine E7 de l'HPV-16 (pVLP-E7). Nous avons notamment montré que l'injection de pVLP-E7 en intradermique (ID) était capable d'induire de bonnes réponses anti-tumorales dans un modèle murin de cancer obtenu en injectant dans le flanc des cellules d'une lignée exprimant les antigènes E6 et E7 de l'HPV-16, mais qu'il était nécessaire d'ajouter des adjuvants de types agoniste de TLR 7 et 9 dans des tumeurs avancées. Afin de tester de nouvelles voies vaccinales dans un modèle pertinent, nous avons développé un modèle orthotopique intrabuccal présentant des caractéristiques anatomiques et inflammatoires plus proches des cancers observés chez l'homme que le modèle ectopique. Dans ce modèle, nous avons testé une voie vaccinale muqueuse intrajugale qui a montré de meilleures réponses T CD8+ spécifiques en comparaison à la voie ID. Nous avons montré que ce type de vaccination en association à des adjuvants, était efficace dans des tumeurs établies, en lien avec une infiltration intratumorale et ganglionnaire de lymphocytes T CD8+ spécifique, permettant également une protection lors de rechallenge tumoral. Cette stratégie apparaît donc prometteuse dans le traitement de ces cancers fréquemment récidivants
Head and neck squamous cell cancer (HNSCC) associated with alcohol and tobacco consumption, and recently with human papillomavirus-16 (HPV-16), have bad prognosis despite current therapies. Development of innovative vaccine strategies and adequate pre-clinical tumor models are required to better evaluate HNSCCs. We developed a DNA vaccination that creates non-infectious virus-like particles, which express HPV-16 E7 oncoprotein (pVLP-E7). Results showed that pVLP-E7 induced an E7-specific immune response in vivo and in vitro. Moreover, using an ectopic model of HNSCC that expresses E6/E7 (TC-1), we found that pVLP-E7 intradermic (ID) immunizations induced anti-tumoral responses at early stages. For larger established tumors, pVLP-E7 vaccines were only efficient when administered with TLR-7 and TLR-9 agonists. In an orthotopic model that shares anatomical and inflammatory features with human HNSCC we observed that intra-cheek (IC) infusion of either TC-1 or NR-S1 cells into mice elicited higher numbers of inflammatory infiltrates in the tumor compared to ectopic models. Using this orthotopic IC model, we found that mucosal IC pVLP-E7 vaccination elicited better vaccine-specific CD8+ T-cell responses than ID administration in naive and tumor-bearing mice. Furthermore, pVLP-E7 IC immunizations in combination with TLR agonists led to rejection of established tumors and long-term protection, both of which were associated with E7-specific CD8+ T cell infiltration in tumors and lymph nodes. Our findings demonstrate that pVLP-E7 IC vaccination with adjuvants is efficient against these tumor models and together provides a valuable therapeutic strategy for HNSCCs

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Fontoura, Paulo Pacheco da. "Terapêuticas antigénio-especificas no tratamento das doenças desmielinizantes: estudos sobre a vacinação com ADN e a descoberta de um novo alvo antigénico". Doctoral thesis, Faculdade de Ciências Médicas. Universidade Nova de Lisboa, 2008. http://hdl.handle.net/10362/5202.

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RESUMO A Esclerose Múltipla (EM) é uma doença desmielinizante crónica do Sistema Nervoso Central (SNC), provocada, em grande parte, por um ataque imuno-mediado contra diversos elementos da bainha de mielina. Dentro dos alvos antigénicos desta resposta autoimune, vários componentes proteicos e lipídicos da mielina têm vindo a ser identificados ao longo dos anos, entre os quais se destacam a proteína básica de mielina(MBP), glicoproteína ligodendrocitária da mielina (MOG), proteína proteolipídica (PLP) e glicoproteína associada à mielina (MAG). Com o desenvolvimento do modelo animal de Encefalomielite Autoimune Experimental (EAE), diversas terapias antigénio-específicas foram desenhadas, baseadas na modificação benéfica da resposta autoimune contra a mielina, tais como a administração de mielina ou seus componentes, os copolímeros terapêuticos, os ligandos peptídeos alterados e, recentemente, a vacinação com ácido desoxirribonucleico (ADN) codificador de proteínas de mielina, integrado em plasmídeos e purificado para administração parentérica. Neste trabalho, apresentamos os resultados de um extenso conjunto de experiências, subordinadas a dois temas fundamentais: 1) avaliação do potencial terapêutico, e dos mecanismos de acção, da vacinação tolerizadora com ADN codificador de proteínas de mielina (MBP, MOG, PLP, MAG) na EAE, e da associação desta vacinação com a administração de ADN de citocinas Th2, ou de oligonucleótidos imunomoduladores; 2) identificação e caracterização da resposta imune contra um novo componente da mielina com potencial antigénico, a proteína inibidora do recrescimento axonal, Nogo-A. No que respeita à vacinação com ADN, os nossos resultados comprovam a eficácia desta terapêutica antigénio-específica na prevenção e tratamento da EAE. Os seus mecanismos de acção incluem, entre outros, a supressão anérgica da proliferação antigénioespecífica dos linfócitos T anti-mielina (no modo de prevenção da doença), o enviesamento Th2 da resposta imune (quando co-administrada com a vacina de ADN codificadora da citocina IL-4, funcionando como terapia génica local), e a redução da diversificação de epítopos da resposta humoral anti-mielina, avaliada através de myelin spotted arrays. A associação das vacinas de ADN com oligonucleótidos imunomoduladores GpG, desenvolvidos para contrariar as sequências CpG imunoestimuladoras presentes no vector de vacinação, levou à melhoria da sua eficácia terapêutica, devida, provavelmente, ao efeito estimulador preferencial dos oligonucleótidos GpG sobre linfócitos Th2 e sobre células reguladoras NK-T. Com base nestes resultados a vacinação com ADN foi desenvolvida para o tratamento da EM em humanos, com ensaios clínicos a decorrerem neste momento. Em relação à proteína Nogo-A, estudos de estrutura primária e de previsão de antigenicidade identificaram a região Nogo-66 como alvo antigénico potencial para a EAE. Nas estirpes de ratinho SJL/J e C57BL/6, fomos capazes de induzir sinais clínicos e histológicos de EAE após imunização com os epítopos encefalitogénicos Nogo1-22, Nogo23- 44 e Nogo45-66, utilizando protocolos de quebra de tolerância imune. Ao mesmo tempo, identificámos e caracterizámos uma resposta linfocitária T específica contra os antigénios contidos na região Nogo-66, e uma resposta linfocitária B com diversificação intra e intermolecular a vários determinantes presentes noutras proteínas da mielina. A transferência adoptiva de linhas celulares Th2 anti-Nogo45-66, levou à melhoria clínica e histológica da EAE em animais recipientes induzidos com outros antigénios de mielina, após migração destas células para o SNC. Estes dados comprovam a importância da Nogo-66 como antigénio na EAE, e a eficácia de terapias antigénio-específicas nela baseadas. No seu conjunto, os nossos resultados confirmam o potencial terapêutico das vacinas de ADN codificadoras de proteínas de mielina, bem como a importância dos encefalitogénios contidos na proteína Nogo-A para a fisiopatologia da EAE e da EM, com eventual relevância para o desenvolvimento de novas terapias antigénio-específicas. O aperfeiçoamento futuro destas terapias poderá levar, eventualmente, a uma capacidade de manipulação da resposta imune que permita o tratamento eficaz das doenças inflamatórias desmielinizantes, como a Esclerose Múltipla. ABSTRACT Multiple Sclerosis (MS) is a chronic demyelinating disease of the Central Nervous System (CNS), caused, mainly, by an immune-mediated attack against several elements of the myelin sheath. Among the antigenic targets for this autoimmune response, several proteic and lipidic myelin components have been identified throughout the years, of which myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipidic protein (PLP), and myelin associated glycoprotein (MAG) are the best characterized. With the development of the animal model for MS, Experimental Autoimmune Encephalomyelitis (EAE), several antigen-specific therapies have been designed, based on beneficial modifications of the autoimmune response against myelin. These have included myelin and myelin component administration, therapeutic copolymers, altered peptide ligands and, more recently, vaccination with myelin-protein encoding deoxyribonucleic acid (DNA), integrated into plasmids and purified for parenteral administration. In this work we present the results of an extensive series of experiments, subordinate to two fundamental areas: 1) evaluating the therapeutic potential, and mechanisms of action, of tolerizing myelin protein (MBP, MOG, PLP, MAG) DNA vaccination in EAE, alone and in association with Th2 cytokine DNA administration, or immunomodulatory oligonucleotides; 2) identifying and characterizing the immuneresponse against a new myelin component with antigenic potential, the axonal regrowth inhibitor Nogo-A. Regarding DNA vaccination, our results prove the efficacy of this antigen-specific therapy for the prevention and treatment of EAE. Its mechanisms of action include, among others, anergic suppression of antigen-specific T-cell proliferation against myelin (in prevention mode), Th2 biasing of the immune response (when co-administered with the IL- 4 codifying DNA vaccine, acting as local gene therapy), and reduction of epitope spreading of the anti-myelin antibody response, assessed by myelin spotted arrays. The combination of myelin DNA vaccination with the administration of GpG immunomodulatory oligonucleotides, designed to counteract immunostimulatory CpG motifs present in the vaccination vector, led to an improvement in therapeutic efficacy, probably due to the preferential stimulatory effect of GpG oligonucleotides on Th2 lymphocytes and on regulatory NK-T cells. Based on these results, tolerizing DNA vaccination is being developed for human use, with ongoing clinical trials. As concerns the Nogo-A protein, based on studies of primary structure and prediction of antigenicity, we identified the Nogo-66 region (responsible for the most of the inhibitory capacity of this protein) as a potential antigenic target for EAE. In the SJL/Jand C57BL/6 mouse strains, we were able to induce clinical and histological signs of EAE,after immunization with the encefalitogenic epitopes Nogo1-22, Nogo23-44 and Nogo45-66,using a tolerance breakdown protocol. Concomitantly, we identified and characterized a specific T cell response against these antigens, together with a B cell response which showed extensive intra and intermolecular epitope spread to several determinants present in other myelin proteins. Adoptive transfer of nti-Nogo45-66 Th2 cell lines resulted in clinical and histological improvement of EAE in recipient animals induced with other myelin antigens, after intraparenchymal CNS migration of anti-Nogo cells. These data confirm the relevance of Nogo-66 as an antigen in EAE, as well as the efficacy of antigenspecific therapies based on the response against this protein.In conclusion, our results substantiate the therapeutic potential of myelin-encoding DNA vaccination, as well as the importance of encefalitogenic epitopes present in the Nogo-A protein for the pathophysiology of EAE and MS, with potential relevance for the creation of new antigen specific-therapies. The future development of these therapies may eventually lead to a degree of manipulation of the immune response that allows the effective treatment of autoimmune, inflammatory, demyelinating diseases, such as Multiple Sclerosis.

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Tyrer, Peter Charles, i n/a. "Targeting M-cells for oral vaccine delivery". University of Canberra. Health Sciences, 2004. http://erl.canberra.edu.au./public/adt-AUC20060427.122012.

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An in vitro model of the follicle-associated epithelia that overlie the Peyer's patches of the small intestine was developed and validated to examine the mechanisms of mucosal antigen sampling. This model displays many phenotypic and physiological characteristics of M cells including apical expression of [alpha]5[beta]l integrin and enhanced energy dependent participate transport. CD4+ T-cells were shown to be an important influence on the development of Mlike cells. The model was used to examine the M cell mediated uptake of several putative whole-cell killed bacterial vaccines. Greater numbers of non-typeable Haemophilus influenzae NTHi 289, NTHi 2019, Escherichia coli 075 HMN and Streptococcus pneumoniae were transported by model M cells compared to control Caco-2 enterocyte-like cells. Studies in isolated murine intestine segments confirmed the selective uptake of NTHi 289 and Escherichia coli demonstrating that intestinal mucosal sampling of these antigens is performed by M cells. Pseudomonas aeruginosa was not absorbed as whole cell bacteria but as soluble antigen, as indicated by the presence of bacterial DNA in the cytoplasm of epithelial cells. These results suggest that bacteria such as NTHi and E. coli are sampled by the mucosal immune system in a different manner to that of bacteria such as Pseudomonas. A number of potential cell surface receptors were investigated to identify which molecules are responsible for intestinal uptake whole-cell killed bacteria. Immunofluorescence studies detected the presence of toll-like receptor-2, toll-like receptor-4, PAF-R and [alpha]5[beta]l integrin on in vitro M-like cell cultures. Examinations of murine intestine confirmed the presence of TLR-4 and PAF-R. TLR-4 was found in small quantities and on M cells. In contrast to the M cell model, TLR-2 expression in the murine intestine was sparse. Receptor inhibition experiments provided evidence for the involvement of TLR-4, PAF-R and [alpha]5[beta]l integrin in M cell uptake of killed bacteria both in vitro and in vivo. This thesis has contributed valuable information regarding the mechanisms of uptake of whole-cell killed bacteria by the intestinal mucosal immune system. For the first time, M cell sampling of whole-cell killed bacteria has been demonstrated. Furthermore, the receptors involved in these processes have been identified. This information will be of great use in the development and optimisation of new oral vaccines.

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Audsley, Jennifer M., i jennifer audsley@med monash edu au. "Alternative Approaches In The Preparation And Growth Of Influenza B Vaccine Viruses". RMIT University. Applied Sciences, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080414.141937.

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Influenza B viruses are a significant cause of disease and influenza B antigens are present in all human vaccines. Achieving suitable yields of seed viruses is often difficult for vaccine manufacturers. With influenza A viruses increases in yields have been achieved by the preparation of reassortants between a high-yielding donor strain and an epidemic strain. However, reassortment of influenza B viruses for the preparation of seeds has not been usually undertaken due to the lack suitable donor strains. Such an approach, which formed the basis of this thesis, could improve vaccine yields, lower costs and introduce a further element of predictability to vaccine manufacture. Potential donor strains were prepared from B/Lee/40 (B/Lee) by two approaches involving the selection of stable cold- and high- temperature mutants. Initial passaging was undertaken in specific-pathogen-free (SPF) chicken embryo kidney (CEK) cultures and later passage in SPF embryonated chicken eggs. Both approaches were successful, although a smaller number of viable progeny could be isolated from plaques obtained at 38aC. Potential donor strains, isolated by selection at either 25 or 38aC and plaque-purified in SPF CEK cultures, were tested for haemagglutinin and infectious titre, in comparison with the original parental strain by three methods, and for differences in antigenicity by cross-haemagglutination-inhibition tests. Potential donor strains selected at temperatures of 25aC (C25) and 38aC (H38) produced haemagglutination titres of 320 units/50ÝL and infectivities of 8.57 and 8.39 50% egg infectious doses, respectively, when grown in eggs at the permissive temperature (34aC). Reassorting experiments using the B/Lee-derived potential donor strains C25 and H38 and the epidemic strain, B/Johannesburg/5/99 (B/Johannesburg), showed that the preparation of reassortant progeny with both epidemic strain HA and NA was difficult. Only 1/24 of the resulting reassortants possessed both the HA and NA of the epidemic strain. None of the reassortant progeny produced in reassorting experiments using C25 and H38 and the epidemic strain B/Panama/45/90 (B/Panama) possessed the desired 6:2 gene constellation (i.e. genes for the two surface antigens of the epidemic strain and the remainder from the donor strain). The infectious titre of selected progeny from the reassortment experiments were determined by three methods and compared with their respective epidemic parents. Yields of several influenza B epidemic strains and potential donor strains were measured after growth in Madin-Darby canine kidney (MDCK) cells prepared in serum-containing (SC) and animal- and human-derived protein-free (AHPF) media. Optimal multiplicities of infection were determined for B/Panama, B/Johannesburg and C25 in MDCK cultures grown in SC medium. A series of experiments were then undertaken to determine the maximum virus yields in MDCK cells grown in SC medium, followed by a further experiment using C25, B/Panama, B/Johannesburg, and selected reassortants after preparation in AHPF medium. Cell culture yields from 5/6 viruses grown in MDCK cells prepared in AHPF medium were higher than in cells prepared in SC medium and approached those obtained in eggs.

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com, movahedi ar@gmail, i Abdolreza Movahedi. "A reverse vaccinology approach to identifying subunit proteins for use in vaccines against Brachyspira pilosicoli infections in humans and animals". Murdoch University, 2008. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20090318.140633.

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The anaerobic intestinal spirochaete Brachyspira pilosicoli is the causative agent of intestinal spirochaetosis (IS), a disease of humans and a number of animal species. IS has been reported in adults and children worldwide but the prevalence in people living in poor hygienic conditions, indigenous populations, homosexual males, and in immunocompromised people is much higher than in other populations. IS is also widespread in pigs and chickens, and causes significant economic impact in the associated industries. To date attempts to develop a vaccine against B. pilosicoli to protect humans and animals have not been successful. In this study a reverse vaccinology approach was used, in which 24 putative open reading frames (ORFs) derived from a partial genome sequence of B. pilosicoli were subjected to in silico and laboratory screening processes to identify potential efficacious vaccine antigens. In silico analysis of the ORFs using a range of bioinformatics algorithms assigned 12 ORF products as periplasmic, outer membrane, or secretory proteins, and these were given a high priority as potential vaccine candidates. The 12 selected ORFs were amplified from a human strain of B. pilosicoli (Wes-B) and cloned. Products from nine ORFS were successfully over-expressed in an Escherichia coli expression system, and then purified using affinity chromatography. In an in vitro immunogenicity trial all the recombinant proteins except for NAV-P27 were strongly recognised in Western immunoblots by a mouse serum raised against B. pilosicoli strain WesB, and by a subset of convalescent sera from pigs naturally and experimentally infected with B. pilosicoli. In an analysis of in vivo immunogenicity, the post-immunisation mouse sera raised against each recombinant protein reacted strongly with each specific proteins, and also recognised the native protein in extracts of B. pilosicoli strain WesB. Sequence analysis of four randomly selected ORFs showed that these were highly conserved amongst the genomes of different human and swine strains of B. pilosicoli. Evaluation of all the data obtained in the reverse vaccinology approach resulted in selection of four ORF products (NAV-P3, NAV-13, NAV-22 and NAV-31) as being potentially protective antigens to be analysed for their further efficacy. These four recombinant proteins were assessed for their efficacy as vaccine components in a mouse model of IS, where the animals were challenged with a human strain of B. pilosicoli. The proteins all induced systemic and local antibody responses, and tended to reduce spirochaete colonisation following experimental infection. These proteins used individually or in combination now have the potential to be further developed into a new vaccine to prevent B. pilosicoli infections.

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Shan, Songhua. "Development and evaluation of DNA vaccines in chickens against a wild bird H6N2 avian influenza virus from Western Australia /". Murdoch University Digital Theses Program, 2009. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20100211.201257.

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Lendemans, Dirk G., i n/a. "Novel cationic preparations of iscoms as vaccine carriers". University of Otago. School of Pharmacy, 2006. http://adt.otago.ac.nz./public/adt-NZDU20060810.141916.

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Aim of thesis: Immuno-stimulating complexes (ISCOMs) are particulate vaccine delivery systems composed of Quillaja saponins, cholesterol and phospholipid. ISCOMs are typically spherical cage-like structures with a diameter of 40 nm and carry a negative charge. Incorporation of the respective vaccine antigen into the particles generates more potent vaccines than a simple mixture of both vaccine components. This requires the antigen to display either hydrophobic domains or positive charges, which allow interaction with the ISCOM particles. However, not all antigens fulfil this requirement and modification of these becomes necessary. Hence, the aim of this study was to design novel preparations of ISCOMs with a positive charge, suitable for adsorption of native hydrophilic antigens and poly-nucleotides, and test their potential as a novel vaccine carrier platform. Methods: Two cationic lipids, DC-cholesterol and DOTAP, were selected to prepare the cationic modifications of ISCOMs. DC-cholesterol substituted for cholesterol in classical ISCOMs, whereas DOTAP substituted for their phospholipid component. The phase behaviour of colloidal systems containing Quil-A, phosphatidylcholine (PC) and DC-cholesterol and of colloidal systems comprised of Quil-A, cholesterol and DOTAP was studied by transmission electron microscopy (TEM). Lipid-film hydration was utilised as the first method to prepare these colloidal systems. Selected compositions containing either DC-cholesterol or DOTAP were also prepared by dialysis as second method. A novel third method for preparing homogenous dispersions of classical ISCOMs was developed utilising ethanol injection. This method was also applied in an attempt to prepare cationic modifications of ISCOMs including DC-cholesterol and DOTAP. As in the colloidal systems comprising Quil-A, PC and DC-cholesterol transformations of structures were observed upon dilution with aqueous solutions, these transitions were also studied on classical ISCOMs using TEM and dynamic light scattering techniques. Loading of cationic colloidal structures composed of Quil-A, PC and DC-cholesterol was performed with the model protein antigen ovalbumin (OVA) and a model plasmid, and the resulting structures were analysed by fluorescence spectroscopy, TEM and gel electrophoresis. The immunological properties of non-loaded and OVA-loaded structures were studied in terms of their ability to activate murine bone marrow derived dendritic cells (mBMDC) as antigen presenting cells (APC) and OVA-specific CD8+ T cells in vitro. Results: Substitution of cholesterol in classical ISCOMs with DC-cholesterol resulted in the formation of cationic cage-like structures similar to the classical particles. These were observed in pseudo-ternary Quil-A:PC:DC-cholesterol systems and even in pseudo-binary Quil-A:DC-cholesterol systems prepared by lipid-film hydration. Compositions at which cage-like structures were observed included high weight proportions of DC-cholesterol (> 60%). However, samples were relatively heterogeneous, and aggregation of colloidal structures was observed at equimolar ratios of Quil-A and DC-cholesterol. The ionic strength, pH and composition of the hydration buffer were demonstrated to be important variables influencing the formation of cage-like structures. Morphological changes of pre-formed cationic cage-like structures were observed upon dilution. However, classical anionic ISCOMs showed a similar behaviour. The numbers of cationic cage-like structures appeared to increase upon prolonged storage of samples. Purification of structures and longitudinal analysis of their composition suggested an increased formation of stoichiometrically defined DC-cholesterol:Quil-A:PC complexes over time, rather than a change in composition. The substitution of phospholipid in classical ISCOMs with DOTAP also resulted in heterogeneous dispersions, and aggregation of colloidal structures was observed at equimolar ratios of Quil-A and DOTAP. Phase separation phenomena were proposed based on TEM observations. However, the formation of cage-like particles with a positive [zeta]-potential was not observed. Although ethanol injection was introduced as a novel method to prepare classical ISCOMs, its application did not result in more homogenous dispersions of cationic colloidal structures containing DC-cholesterol or DOTAP. Dialysis also failed to produce higher numbers of well-defined cationic particles, although using this method homogeneous, anionic ISCOM-like particles containing DOTAP were obtained. The efficient adsorption of OVA and plasmid DNA onto cationic structures containing Quil-A, PC and DC-cholesterol was demonstrated. The adsorption process was accompanied with a decrease in [zeta]-potential, aggregation of structures and changes in the ultra-structure, particularly at high protein:lipid ratios. The in vitro immunogenicity of dispersions containing Quil-A, PC and DC-cholesterol was equivalent to that of classical ISCOMs in terms of activation of mBMDC and OVA-specific CD8+ T cells, even though smaller amounts of Quillaja saponins and total lipid were co-delivered with OVA. Furthermore, the uptake of OVA by BMDC appeared to be more efficient in conjunction with the novel cationic dispersions. Conclusions: Cationic colloidal structures containing Quillaja saponins offer great potential as vaccine delivery systems. Their advantages thus far include simple and efficient adsorption of antigen, efficient uptake by APC and immunological activity in vitro. With further development, cationic carriers containing Quillaja saponins may constitute a very potent vaccine delivery platform suitable for a variety of subunit antigens, and suffice both pharmaceutical and immunological requirements.

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White, Karen Louise, i n/a. "Modified liposomes as adjuvants". University of Otago. School of Pharmacy, 2005. http://adt.otago.ac.nz./public/adt-NZDU20070126.131417.

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Despite the progress in elucidating antigens for both therapeutic and prophylactic vaccines, safety concerns over current vaccine delivery vehicles and adjuvants has limited the development of new vaccines. In particular, there is an urgent need for effective vaccines capable of stimulating cytotoxic T lymphocyte (CTL) responses against intracellular pathogens or tumor cells. Liposomes are under investigation as a particulate vaccine delivery system with the required safety profile and demonstrated ability to target antigens to dendritic cells (DC), the cells of the immune system responsible for initiating effective and long lasting CTL immune responses. Unmodified liposomes however, are inherently non-immunogenic and thus not capable of stimulating activation of DC, which is a necessary step in immune activation. In this thesis the use of modified liposomes to more efficiently target vaccine antigens to DC and then activate the DC sufficiently to initiate down-stream immune responses was investigated. In the first approach to liposome modification, mannosylated phospholipids were incorporated within the liposome bilayer to target C-type lectins on DC. Incorporation of mono- or tri-mannosylated phospholipids within liposomes was found to be an effective means of attaching mannose-containing ligands to the liposome surface without compromising the integrity of the liposome structure. The uptake of tri-mannose-containing liposomes was enhanced in human monocyte derived DC (MoDC) compared to both unmodified liposomes and mono-mannose-containing liposomes. In contrast, neither mono- nor tri-mannose-containing liposomes were taken up by murine bone marrow derived DC (BMDC) to a greater extent than unmodified liposomes. This finding may reflect the differences in ligand specificity for C-type lectins on DC derived from different mammalian species. It was also found in these studies that increased uptake of liposomal antigens by DC does not necessarily result in increased DC activation, as evidenced by a lack of up-regulation of DC surface activation markers and ability to stimulate T cell proliferation. The second approach to liposome modification involved the incorporation of lipid core peptides (LCPs) into the liposome structure. LCPs alone were demonstrated to be able to stimulate DC and subsequent CD8+ T cell activation in vitro. LCP-based vaccines were also able to stimulate effective cytotoxic immune responses in vivo, and protect against tumor challenge, but only if administered in alum with CD4 help. Liposomes containing LCPs were able to stimulate greater DC activation and subsequent CD8+ T cell proliferation in vitro compared with unmodified liposomes. In the in vivo studies however, LCP-containing liposomes were not able to stimulate a cytotoxic immune response or protect against tumor challenge as effectively as LCP administered in alum. In the final approach to liposome modification, inclusion of the adjuvant Quil A was investigated for its ability to increase the immunogenicity of LCP-containing liposomes. It was found that small amounts of Quil A could be incorporated into liposomes without compromising the liposome bilayer. The inclusion of as little as 2% Quil A was able to stimulate DC activation and subsequent T cell proliferation in in vitro studies. In addition, immunisation of mice with LCP-containing liposomes with incorporated Quil A was found to stimulate an in vivo CTL immune response comparable to LCPs administered under optimal vaccine conditions. In conclusion, the work presented in this thesis demonstrates that modified liposomes are a useful vaccine delivery system for the initiation of in vivo cytotoxic and prophylactic immune responses.

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com, tickle_me_patty@hotmail, i Patrick Leslie Shearer. "Development of Novel Diagnostic and Vaccine Options for Beak and Feather Disease Virus". Murdoch University, 2009. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20090720.142800.

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Beak and Feather Disease Virus (BFDV) is a circovirus which causes ill-thrift, feather loss and immunosuppression leading to secondary infections and eventually death in psittacine birds. The development of standardised reagents for the detection and characterisation of BFDV infections and for the production of protective vaccines has been difficult as no cell culture system has yet been found to grow the virus successfully in vitro. However, the development of consistent and effective diagnostic tests and vaccines is now more practical through the application of nucleic acid-based detection methods and recombinant technology. A quantitative real-time PCR assay for the detection of BFDV DNA was developed, using primers designed to amplify a conserved 81 bp fragment of ORFV1 and SYTO9, a fluorescent intercalating dye, with assays run on a Corbett RotorGene 3000. A synthetic oligonucleotide was used to establish standard curves for the quantitation of viral load in both blood and feather preparations. The assay was very sensitive, with a detection limit of 50 copies/ìL. The assay was developed using BFDV-positive DNA extracts from the feathers of 10 different species of birds and validated with blood and feather samples from corellas vaccinated with an experimental BFDV vaccine, then challenged with live virus. Viral DNA was reliably detected in the blood of all control (non-vaccinated) birds and in some vaccinated birds. Contamination of the environment with the feather dander of BFDV-infected birds meant that HA feather preparations were unreliable for the detection and quantitation of viral excretion. Nonetheless, the assay should prove to be a useful and sensitive test for the detection of viral DNA in a range of samples in future investigations. A recombinant BFDV capsid protein was also produced and a specific monoclonal antibody developed against it. The behaviour of the protein in haemagglutination (HA) assays and the behaviour of the monoclonal antibody in western blotting, immunohistochemistry (IHC), ELISA and haemagglutination-inhibition (HI) assays were characterised. The protein had the ability to agglutinate galah erythrocytes as per the wild-type virus and this agglutination was successfully inhibited by antibodies to wild-type BFDV from naturally immune psittacine birds. Furthermore, the protein self-assembled into virus-like particles as determined by electron microscopy. The antibody was specific for both the recombinant BFDV capsid protein and the whole virus and had similar optimal titres when used in western blotting and IHC. The antibody also had HI activity and detected BFDV virus from 3 genera of psittacine birds, including the recently described cockatiel BFDV isolate. A novel blocking (or competitive) ELISA (bELISA) for the detection of anti- BFDV antibodies in psittacine sera (Ab-bELISA) was also developed and validated with 166 samples from eastern long-billed corellas vaccinated with the recombinant capsid protein and challenged with live virus. The bELISA was found to be both sensitive and specific and correlated strongly with the HI test, thus it should have wide application for the serodiagnosis of BFDV. A survey of cockatiels (n=88) housed at commercial aviaries was conducted to investigate whether BFDV infection occurs in cockatiels. All birds were diagnosed as being virus-free by PCR and HA and had no detectable antibody titre by HI assay. In addition to this, the genomes of two BFDV isolates obtained from diseased cockatiel feathers were sequenced and cross-reactivity assays performed using virus eluted from these feathers and sera from naturally immune psittacine birds. Serological cross-reactivity results and phylogenetic analysis of the nucleotide sequences indicated that the cockatiel virus isolates were serologically and genetically different to other BFDV isolates. This is the first report of an antigenically distinct BFDV in psittacine birds. Since the Ab-bELISA has a lower limit of detection than the HI assay, it was used to repeat the cockatiel sero-survey. No antibodies were detectable in any of the cockatiels tested and thus questions about the real prevalence of BFDV infection in cockatiels and the possible existence of a novel BFDV serotype adapted to cockatiels remain unanswered. The successful control of PBFD in both pet and wild birds depends on the development of vaccines that incite a strong specific immune response and can be efficiently produced in large quantities. Recombinant BFDV capsid proteins have recently been considered as candidate vaccines against BFDV and recombinant techniques allow the development of other candidate vaccines, including DNA vaccines. In order to examine the potential of DNA vaccination as a strategy for the prevention and control of BFDV, two DNA vaccines, based on the nucleotide sequence encoding the capsid protein of BFDV, were developed using the mammalian expression vector pVAX1. The vaccine constructs encoding both the full length and NLS-truncated capsid protein resulted in protein expression both in vitro and in vivo. Protein was detected in COS-7 cells transfected with the constructs with an indirect immunocytochemistry assay using the monoclonal antibody described in Chapter 5. Protein was present in the nucleus of cells transfected with the vaccine encoding the full-length nucleotide sequence and in the cytoplasm of cells transfected with the vaccine encoding the NLS-truncated sequence as expected. Both DNA vaccine constructs induced detectable levels of anti-BFDV antibodies in vaccinated birds, determined using the Ab-bELISA described in Chapter 5. Thus, DNA vaccines similar to those presented here may have application in the prevention and control of BFDV and some options for the further development of these vaccines into effective methods for the control of BFDV are discussed.

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Lauer, Katharina. "A multipathogen vaccine for rabies, hepatitis B, Japanese encephalitis and enterovirus 71". Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/a-multipathogen-vaccine-for-rabies-hepatitis-b-japanese-encephalitis-and-enterovirus-71(f489f961-317e-4430-becc-0474cae79268).html.

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To enhance the global control of encephalitis and hepatitis caused by rabies virus (RABV), Japanese encephalitis virus (JEV), enterovirus 71 (EV71) and hepatitis B virus (HBV), novel immunisation strategies are needed. All four diseases particularly affect low income countries with marginal health services – an affordable combined vaccine strategy could alleviate the large burden of disease. Therefore, we aimed to construct a multipathogen vaccine assessing the immunising activity of a recombinant modified vaccinia Ankara (MVA), expressing key antigens (RABV-glycoprotein, JEV pre-membrane & envelope protein, EV71-P1 protein and large hepatitis B surface antigen) from the various pathogens. Successful delivery of the pathogen sequences into non-essential sites (deletion site I, II, VI) of MVA via homologous recombination with a transfer plasmid, was demonstrated by transient color selection (LacZ-marker) in vitro. The stable insertion of the expression cassettes was validated over ten virus passages by PCR with specific primer sets, targeting the pathogen sequence. Two recombinants, one carrying the EV71 and JEV pathogen sequence and one carrying the RABV-HBV pathogen sequence were generated and validated by PCR.To ensure similar expression of the key antigens, a T7-promoter was linked to the expression cassettes of all pathogen sequences. Direct regulation of this promoter was achieved through co-infection with a second T7-polymerase expressing MVA under the control of a vaccinia p7.5 promoter. Protein expression from recombinant MVA using the co-infection model of expression in vitro, was further characterised by Western blot, dot blot and immunocytochemistry. All inserted transgenes were expressed using an avian (chicken embryo fibroblasts) or mammalian (human fetal lung fibroblasts) cell culture system. To investigate the co-infection model of antigen delivery in vivo, a pilot murine immunogenicity study was performed in six Balb/c mice using the MVA-RABV-HBV recombinant in a homologous prime-boost regimen two weeks apart. To detect antibodies against the expressed pathogen sequences in the mouse serum an antibody-capture assay was performed (Western blot, dot blot). The antigen (used to capture murine antibodies) was purified RABV-glycoprotein or large hepatitis B surface antigen expressed from a baculovirus. The murine antibodies were detected by a secondary anti-mouse antibody, conjugated with horseradish peroxidase for a chemiluminescent reporter assay. Although, serum antibodies against MVA were induced in all mice, no serum antibodies against RABV or HBV could be detected. In summary, we were able to demonstrate that two transgenes, when inserted into one or two different loci in the MVA genome, can be expressed in vitro when using the co-infection model of gene expression with a T7-expression system. This project has provided new insights into a novel group of vaccines, the multipathogen viral vector vaccines, employing MVA as a vector. Future studies will be needed to further explore this vaccine-group, as well as the co-infection model of expression.

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Dalba, Charlotte. "Nouveaux vecteurs rétroviraux pour l'immunothérapie des cancers et des infections virales". Paris 6, 2006. http://www.theses.fr/2006PA066604.

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Les vecteurs rétroviraux défectifs pour la réplication (RDR) dérivés des oncorétrovirus murin ont marqué l’histoire du fait de leur utilisation pour les premières thérapies géniques réussies. Malgré cela, ces vecteurs présentent deux désavantages majeurs : une efficacité de transfert de gène in vivo insuffisante et une mutagénicité insertionnelle. Notre travail a consisté à surmonter ces problèmes lors de la génération de vecteurs destinés à des applications dans les domaines du cancer et des maladies infectieuses. Ceci nous a amené à concevoir, réaliser et évaluer deux types de vecteurs ayant des caractéristiques opposées : (i) des vecteurs rétroviraux compétent pour la réplication (RCR) et (ii) des pseudo-particules rétrovirales (VLP) dépourvues de génome. Notre travail illustre la versatilité des vecteurs rétroviraux, qui permet des designs novateurs et en font des acteurs majeurs de la thérapie génique et des vaccins génétiques
Replication defective retroviral (RDR) vectors based on murine oncoretroviruses marked history by being the gene transfer vectors used in the first successful gene therapies. Despite this achievement, they have two major drawbacks: insufficient efficacy for in vivo gene transfer and insertional mutagenesis. During the course of the work presented here, we attempted to overcome these problems while making vectors useful for two different indications; cancer and infectious disease. This led to the conceptualization, realization and evaluation of two retroviral vector designs of principally opposite character; (i) replication competent retroviral (RCR) vectors transducing largely complete genomes and (ii) genome free virus-like particle (VLP) vectors. Our work illustrates that the versatility of retroviral vectors, which permits inventive designs, make them major actors in gene therapy and genetic vaccines

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Holden, James Anthony, i jamesholden@netspace net au. "Vaccination Strategies for the Prevention of Swine Dysentery". RMIT University. Applied Sciences, 2006. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20070112.122102.

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The SmpA outer membrane lipoprotein of B. hyodysenteriae has several characteristics that indicate the potential to protect against swine dysentery (SD). It localises to the outer membrane and antibodies directed against SmpA can prevent the growth of B. hyodysenteriae in vitro. There is some variation observed in the distribution and expression of the SmpA lipoprotein, suggesting that vaccination with SmpA may not provide protection against challenge with a heterologous B. hyodysenteriae strain. This study has characterised the variation at the smpA locus, and in the process has identified a novel gene, smpB. There is very low similarity between smpB and smpA, with the exception of an identical lipoprotein signal sequence. This suggests that SmpB may be translocated to the outer membrane of B. hyodysenteriae in a similar fashion to SmpA. The results described in this thesis indicate that strains of B. hyodysenteriae harbour either smpA or smpB, but not both, explaining the earlier results of Turner et al. (1991). The presumed outer membrane location of SmpB lead to further investigations into its potential to protect mice from infection with B. hyodysenteriae. Swine Dysentery is a inflammatory disease of the swine colon. Therefore it is believed that a mucosal immune response may provide increased protection against challenge. In this study, vaccination of mice with recombinant SmpB elicited high levels of serum antibodies, induced the production of Interleukin-4 producing T lymphocytes and decreased the observed histological effects after challenge with virulent B. hyodysenteriae. In efforts to increase the protected conferred by vaccination with SmpB, recombinant Salmonella typhimurium STM-1 vaccines were created to express SmpB or deliver DNA vaccines encoding SmpB. Vaccination with these recombinant Salmonella vectors did not induce a measurable SmpB specific immune response. Macrophage survival and plasmid stability studies indicated that this was due to instability of the expression plasmids in STM-1. Although SmpB will only ever protect against strains of B. hyodysenteriae harbouring smpB, these results indicate that with further research, SmpB (and SmpA) may contribute to protection from SD. Toxin production is an important aspect of the pathogenesis of many pathogenic bacteria. Vaccination with attenuated toxins is commonly used to prevent disease. In this study, the B. hyodysenteriae â-haemolysin HlyA was used to vaccinate mice to determine the protection induced after challenge. Vaccination of mice with recombinant HlyA induced significant levels of serum antibodies and lowered the observed pathological effects after challenge of vaccinated mice with virulent B. hyodysenteriae. In an attempt to increase the mucosal immune response and therefore the protection afforded after vaccination with HlyA, recombinant S. typhimurium STM-1 strains were created to express HlyA or deliver DNA vaccines encoding HlyA. Similar to the recombinant STM-1 vaccines expressing SmpB, a HlyA specific immune response was not observed by ELISA or ELISPOT analysis. Plasmid stability trials revealed that the inability to induce a detectable HlyA specific immune response by recombinant STM-1 vaccination may be due to ins tability of the plasmids. Outer membrane proteins are often important components of vaccines against bacterial and viral pathogens. Considering the variation observed in the smpA locus in this study resulting in the identification of smpB, further investigation into the distribution and conservation of outer membrane encoding genes in B. hyodysenteriae strains was undertaken. In particular, the blpAEFG, vspABCD and vspEFGH clusters were analysed for their distribution. It was demonstrated that genes that are B. hyodysenteriae specific (vspABCD and vspEFGH) displayed higher levels of polymorphism than those that are distributed amongst non-pathogenic species, such as B. innocens (which contains blpAEFG). This suggests that the variation in the vspABCD and vspEFGH clusters amongst B. hyodysenteriae strains may be a result of the exposure to the host immune system. Further investigation was undertaken by PFGE analysis and 2D-gel electrophoresis, to analyse genomic and proteomic variation at a global level. Although strains of B. hyodyse nteriae produced several different electrophoretic types (ET) upon PFGE analysis, only limited correlation between the PFGE ET, the polymorphisms in vspABCD and vspEFGH and the presence of smpA/smpB were observed. 2D-gel electrophoresis analysis of outer membrane preparations of two B. hyodysenteriae strain revealed several distinct differences in the outer membrane between B. hyodysenteriae strains. The observed differences in the proteins contained in the outer membrane of B. hyodysenteriae is important for vaccine design, as the induction of cross protection between strains of B. hyodysenteriae is essential for a effective vaccine.

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au, T. La@murdoch edu, i Tom La. "Characterisation, Recombinant Expression and Immunogenicity of BHLP29.7, An Outer Membrane Lipoprotein of Brachyspira Hyodysenteriae". Murdoch University, 2006. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20070830.141953.

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Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study, the gene encoding a 29.7 kDa outer membrane lipoprotein of the causative intestinal spirochaete Brachyspira hyodysenteriae, was identified and sequenced. An 816 bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site, and putative 10 and 35 promoter regions upstream from the start of the ORF. The 29.7 kDa outer membrane lipoprotein was designated Bhlp29.7 and the encoding gene named bhlp29.7. The amino acid sequence of Bhlp29.7 included a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. In silico analysis of this protein together with lipidation studies further supported its probable outer membrane localisation. Comparison of the Bhlp29.7 sequence with public sequence databases showed that it had up to 40% similarity with the D-methionine substrate-binding outer membrane lipoprotein (MetQ) of a number of bacterial pathogens. The Bhlp29.7 gene was detected in all 48 strains of B. hyodysenteriae examined, and in Brachyspira innocens strain B256T, but not in 10 other strains of B. innocens or in 42 strains of other Brachyspira spp. The gene was sequenced from B. innocens strain B256T and from 11 strains of B. hyodysenteriae. The B. hyodysenteriae genes shared 97.9-100% nucleotide sequence identity and had 97.5-99.5% identity with the gene of B. innocens strain B256T. The Bhlp29.7 gene was subsequently cloned and expressed as a histidine fusion protein in an Escherichia coli expression system. An ELISA test using recombinant his-tagged Bhlp29.7 (His6-Bhlp29.7) as the detecting antigen was developed and evaluated. The threshold value of the test was chosen to provide a highly stringent assessment of the disease status of a herd. The sensitivity and specificity of the test was 100%. When the test was applied to sera from eight herds with suspected SD, four gave ELISA values indicating that the herds were diseased. The remaining four herds gave ELISA values below the threshold value. These results indicated that the Bhlp29.7-ELISA was useful as an indirect test for exposure of a herd to B. hyodysenteriae and may be a helpful complement to current methods of SD diagnosis. Recombinant His6-Bhlp29.7 was evaluated as a vaccine subunit for prevention of SD. The His6-Bhlp29.7 was shown to be immunogenic in mice following two intramuscular injections. Vaccination of mice with His6-Bhlp29.7 provided full protection after oral challenge with B. hyodysenteriae. In two experiments, intramuscular and oral vaccination of pigs with the His6-Bhlp29.7 resulted in a 50% reduction in incidence of SD compared to unvaccinated control pigs (P=0.047). This is the first subunit vaccine shown to provide pigs with protection from SD. Further work is needed to optimise delivery routes and adjuvants for commercial development of the vaccine.

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Giles, Natalie Lydia. "Exploitation of the protein tubulin for controlling African trypanosomiasis /". Access via Murdoch University Digital Theses Project, 2005. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20060315.191003.

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Shearer, Patrick. "Development of novel diagnostic and vaccine options for beak and feather disease virus (BFDV) /". Murdoch University Digital Theses Program, 2008. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20090720.142800.

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Murdoch University (Ph.D.)--Murdoch University, 2008.
Contains three published journal articles at back of thesis. Thesis submitted to the Faculty of Health Sciences. Includes bibliographical references (leaves 196-231)

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