Tesis: "Immunogeni"

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Sukkurwala, Abdul Qader. "Autophagy : A New Modulator of Immunogenic Cell Death for Cancer Therapy". Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T031.

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Certains agents chimiothérapeutiques tels que les anthracyclines ou l'oxaliplatine induisent une mort cellulaire immunogène, ce qui implique que les cellules mourrantes du patient servent de vaccin thérapeutique en stimulant une réponse immunitaire antitumorale. La mort cellulaire immunogène est caractérisée par la libération de signaux d'alarme par la cellule tumorale mourante qui permettent l’activation du système immunitaire. En premier lieu, l'exposition de la calréticuline à la surface de la cellule tumorale mourante va agir comme un signal de type «eat-me» pour les cellules dendritiques. Une fois relâchée, la protéine nucléaire HMGB1 se lie au récepteur TLR4 afin de faciliter la présentation antigénique. Les cellules mourantes vont également libérer de l'ATP qui agit sur les récepteurs P2X7 et active l’inflammasomme NLRP3, conduisant à la libération d'IL-1β et ainsi à l’activation des cellules T CD8+ productrices d’IFN-γ. L’autophagie est un mécanisme cellulaire qui est activé en réponse à la chimiothérapie. L'autophagie signifie «self-ating», il s'agit d'un processus cellulaire activé par diverses conditions de stress, par lequel les cellules peuvent dégrader les protéines et les organites. Il peut aussi être induit par un stress du réticulum endoplasmique. Ce dernier étant également impliqué dans l'exposition de la calréticuline pendant la mort cellulaire immunogène, nous avons au cours de cette étude cherché à déterminer le rôle de l'autophagie dans la mort cellulaire immunogène. Nous avons constaté que l'autophagie est nécessaire pour la libération de l'ATP après un traitement par des chimiothérapies immunogènes, en observant que le nockdown de gènes essentiels de l'autophagie limitait la sécrétion d'ATP. Nous avons également observé que des cellules déficientes pour l'autophagie traitées par une chimiothérapie immunogène sont incapables d’immuniser des souris contre une injection de cellules vivantes. En outre, les tumeurs déficientes pour l’autophagie ne répondent pas à un traitement systémique immunogène dans des souris immunocompétentes et continuent à proliférer en comparaison à des tumeurs “wild-type”. De plus, nous avons montré que les cellules déficientes pour l'autophagie ne sont pas en mesure de recruter des cellules dendritiques dans le lit tumoral ou d'induire l’activation des cellules T CD8+. A l'inverse, l'inhibition des enzymes de dégradation de l’ATP extracellulaire accroit les concentrations d'ATP dans les tumeurs déficientes pour l'autophagie, ce qui rétablit le recrutement des cellules immunitaires dans le lit tumoral et restaure la réponse chimiothérapeutique des cancers déficients pour l'autophagie. Ainsi, cette étude a montré l'importance de l'autophagie dans la réponse anti-tumorale spécifique, après traitement par des chimiothérapies immunogènes. Ces résultats ouvrent de nouvelles perspectives dans le concept de la mort cellulaire immunogène
In recent years it has been demonstrated that some chemotherapeutic agents such as anthracyclines or oxaliplatin can induce a type of tumor cell death that is immunogenic, implying that the patient’s dying cancer cells serve as a therapeuticvaccine that stimulates an antitumor immune response, which in turn can control or eradicate residual cancer cells. Immunogenic cell death is characterized by the emission of danger signals from the dying tumor cell, which activate the immune system. At first the exposure of calreticulin, acts as an «eat-me» signal for dendritic cells (DCs). Once released, the nuclear protein HMGB1 binds to TLR4 on DCs, facilitating antigen processing and presentation. The dying tumor cells also releases ATP, which acts on P2X7 receptors on DCs and activates the NLRP3 inflammasome, leading to IL-1β release, necessary for IFN-γ-producing CD8+ T cell activation. Autophagy literally ‘self-eating’ is a cellular process activated in response to various conditions of cellular stress, whereby cells can liberate energy resources via the degradation of proteins and organelles. Recently autophagy has been found activated in response to chemotherapy and in this project we aimed to determine the potential role of autophagy in immunogenic cell death. We found that autophagy isrequired for the release of ATP in response to immunochemotherapeutic treatment, as we observed that the knockdown of essential autophagy-related genes abolished its secretion. We observed that autophagy deficient cells treated with immunogenic cell death inducers failed to immunize mice against a re-challenge with living cells. Furthermore, autophagy deficient tumors growing on immunocompetent mice did not respond to systemic immunogenic treatment and continued proliferating in contrast to autophagy proficient tumors. We showed that autophagy deficient cells were neither able to recruit DCs into the tumor bed nor to activate CD8+ T cells. Conversely, the inhibition of extracellular ATP degrading enzymes increased extracellular ATP concentrations in autophagy deficient tumors, which reestablished the recruitment of immune cells into the tumor bed, and restored chemotherapeutic responses in autophagy-deficient cancers. Altogether, this study showed the importance of autophagy in tumor-specific immune response after treatment with chemotherapy, thus giving new insights into the concept of immunogenic cell death

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2

Menger, Laurie Colombe Aude. "Effets anticancereux des glucosides cardiotoniques par induction d'une mort cellulaire immunogène". Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00757180.

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L'efficacité de certains agents anti-cancéreux, notamment les anthracyclines et l'oxaliplatine repose sur l'induction d'une mort cellulaire immunogène (MCI) pouvant conduire à une réponse immunitaire anti-tumorale spécifique. Les cellules succombant à ce type particulier d'apoptose vont subir certaines modifications définies par un modèle spatio-temporel précis. Celui-ci est caractérisé par la mise en place de signaux d'apparition séquentielle, dont le plus précoce est l'exposition membranaire d'une protéine du réticulum endoplasmique, la calréticuline (CRT) qui constitue un signal de danger essentiel à la phagocytose des cellules mourantes par les cellules dendritiques. Ensuite, à un stade apoptotique, la sécrétion d'adénosine triphosphate (ATP) dépendante de l'autophagie active l'inflammasome NLRP3 et induit la polarisation des cellules T CD8+ productrices d'IFN-. Enfin, au cours de la nécrose secondaire, le relargage d'un facteur pro-immunogène High-mobility group protein B1 (HMGB1) est indispensable à une présentation antigénique optimale aux cellules T CD4+ et CD8+, contribuant ainsi à l'activité tumoricide de la chimiothérapie et protégeant l'hôte d'une éventuelle rechute. De manière à identifier de nouvelles molécules capables d'induire une réponse immunitaire anti-tumorale spécifique, un criblage à haut débit de bibliothèques de composés approuvés par la FDA (Food and Drug Administration) a été réalisé grâce à l'utilisation de microscopie automatisée et de biosenseurs permettant la détection de l'exposition de la CRT, de la sécrétion d'ATP et du relargage d'HMGB1. Ce criblage multiparamétrique à haut débit a permis d'identifier les glucosides cardiotoniques (GCs), déjà bien connus pour leur activité cytotoxique préférentielle des cellules cancéreuses, comme étant des inducteurs efficaces de la MCI. Cette découverte a été validée par des méthodes alternatives in vitro, suivis d'une étude de la mécanistique d'induction de la MCI par les GCs. Les résultats ont mis en évidence une inhibition spécifique de la sous-unité α1 de la pompe Na + / K + ATPase, qui à son tour modifie l'homéostasie calcique de la cellule cible, un effet reproduit par les ionophores du Ca2 +. Nous avons ensuite montré que les CGs, en combinaison avec des chimiothérapies non immunogènes (cisplatine ou mitomycine C) pouvaient vacciner des souris syngéniques contre une ré-injection de cellules cancéreuses vivantes et que les effets antinéoplastiques de ces agents endommageants l'ADN pouvaient être potentialisés par les GCs dans les hôtes immunocompétents mais pas dans les souris immunodéficientes. Enfin, une analyse rétrospective de patients atteints de carcinomes et traités par un GC couramment utilisé en clinique dans la prise en charge de l'insuffisance cardiaque, la digoxine (n=145) a révélé une amélioration significative de la survie globale par rapport à celle de patients non traités (n=290). Les patients ont été appariés en fonction de leur âge, sexe, type de cancer et principaux paramètres pronostiques. Des analyses plus approfondies ont ensuite révélées que la digoxine n'affectait pas la survie globale des patients déjà traités par des agents chimiothérapeutiques immunogènes mais celle des patients ayant reçu des agents autres que les anthracyclines ou l'oxaliplatine.

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3

Zhou, Heng. "Mode d'action des composés induits Lytix sur la mort cellulaire". Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS134.

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Le peptide oncolytique LTX-315 a été développé comme un peptide cationique amphipathique qui tue les cellules cancéreuses et qui se révèle stimulant pour la réponse anti-cancer immune quand il est localement injecté dans des tumeurs présentent chez des souris immunocompétentes. La microscopie électronique par transmission révélées que LTX-315 a échoué à induire la condensation nucléaire apoptotique mais induit plutôt un phénotype nécrotique. Par conséquent, LTX-315 a échoué à stimuler l'activation de caspase-3, et l'inhibition des caspases par le biais de Z-VAD-fmk n'a pas été capable de réduire la mort cellulaire par LTX-315. En outre, deux inhibiteurs importants de la nécroptose, nommés necrostatin-1 et cycospotin-A, ont échoué à réduire la mort cellulaire par LTX-315. En conclusion, il semble que LTX-315 déclenche une nécrose non-régulée, qui peut contribuer à ses effets pro-inflammatoires et pro-immunitaires. Le fractionnement subcellulaire des cellules traitées par LTX-315, suivie par une quantification spectrométrique massive, a révélé que cet agent était enrichi en mitochondries. LTX-315 a causé un arrêt immédiat de la respiration mitochondriale sans aucun effet majeur de découplage. Par conséquent, LTX-315 a interrompu le réseau mitochondrial, a dissipé le potentiel mitochondrial de la membrane interne, et a causé la libération des protéines inter-membranaires mitochondriales dans le cytosol. LTX-315 était relativement inefficace dans la mitophagie stimulante. Les cellules dépourvues de deux pro-apoptotiques protéines à domaines multiples BAX et BAK, étaient moins susceptibles de mourir par LTX-315. De plus, les cellules conçues pour perdre leurs mitochondries étaient relativement résistantes à LTX-315, soulignant l'importance de cet organite pour la cytotoxicité induite par LTX-315. Au total, ces résultats soutiennent la notion que LTX-315 tue les cellules cancéreuses par sa vertue à perméabiliser les membranes mitochondriales. Nous avons observé que LTX-315 induit toutes les charactéristiques d'ICD connues. Cette conclusion a étévalidée par diverses méthodes indépendantes incluant la teinture par immunofluorence, dosage par bioluminescence, immunodosages, RT-PCRs. Quand il a été injecté dans des cancers créés, LTX-315 a causé une nécrose focale transitoirement hémorragique accompagnée d'une libération massive de HMGB1, aussi bien que l'activation de caspase-3 dans une partie des cellules. LTX-315 était au moins aussi efficace que le controle positif, l' anthracycline mitoxantrone en induisant une inflammation locale par infitration de cellules myéloïdes et de lymphocytes T. Collectivement, ces résultats appuient l'idée que LTX-315 peut induire l'ICD, expliquant sa capacité à induire des effets thérapeutiques immuno-dépendants.L'autre Lytix composé LTX-401 est un acide aminé oncolytique dérivé avec des propriétés immunogéniques potentielles. Nous démontrons que LTX-401 détruit sélectivement la strucutre du dispositif Golgi. Le fractionnement subcellulaire suivi par une détection spectométrique massive a révélé que LTX-401 a enrichi sélectivement dans le Golgi mieux que dans la mitochondrie ou dans le cytosol. Lagent Golgi-dissociant Brefeldin A a réduit la mort cellulaire par LTX-401 comme cela a partiellement inhibé la libération mitochondrial induite par LTX-401 de cytochrome C et l'activation de BAX. L'effet cytotoxique de LTX-401 a été atténué par la double suppression de BAX et BAK, comme la déplétion mitophagique forcée de la mitochondrie, déjà réfractaire à l'inhibition de caspase. LTX-401 induit toutes les caractéristiques majeures de la mort cellulaire immunogénique. En outre, les tumeurs traitées par LTX-401 ont manifesté une forte infiltration lymphoïde. Ensemble, ces résultats soutiennent l'idée que LTX-401 peut stimuler la mort immunogétique des cellules à travers un passage dans lequel LTX-401 localisé sur Golgi opère en amont de la perméabilisation de la membrane mitochondriale
The oncolytic peptide LTX-315 has been developed as an amphipathic cationic peptide that kills cancer cells and turned out to stimulate anticancer immune responses when locally injected into tumors established in immunocompetent mice. We investigated whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3. Moreover, inhibition of caspases by Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, two prominent inhibitors of necroptosis, necrostatin-1 and cyclosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects. Subsequently, we investigated the putative involvement of mitochondria in the cytotoxic action of LTX-315. Subcellular fractionation of LTX-315-treated cells, followed by mass spectrometric quantification, revealed that the agent was enriched in mitochondria. LTX-315 caused an immediate arrest of mitochondrial respiration without any major uncoupling effect. Accordingly, LTX-315 disrupted the mitochondrial network, dissipated the mitochondrial inner transmembrane potential, and caused the release of mitochondrial intermembrane proteins into the cytosol. LTX-315 was relatively inefficient in stimulating mitophagy. Cells lacking the two pro-apoptotic multidomain proteins BAX and BAK, were less susceptible to LTX-315-mediated killing. Moreover, cells engineered to lose their mitochondria were relatively resistant against LTX-315, underscoring the importance of this organelle for LTX-315-mediated cytotoxicity. Altogether, these results support the notion that LTX-315 kills cancer cells by virtue of its capacity to permeabilize mitochondrial membranes. Following, we investigated whether LTX-315 may elicit the hallmarks of immunogenic cell death (ICD). Overally, we observed that LTX-315 induces all known ICD characteristics. This conclusion was validated by several independent methods including immunofluorescence staining, bioluminescence assays, immunoassays, and RT-PCRs. The injection of LTX-315 into established cancers resulted in transiently hemorrhagic focal necrosis that was accompanied by massive release of HMGB1, as well as caspase-3 activation in a fraction of the cells. LTX-315 was equal or more efficient as the positive control, the anthracycline mitoxantrone (MTX), in inducing local inflammation with infiltration by myeloid cells and T lymphocytes. Collectively, these results support the idea that LTX-315 can induce ICD, explaining its capacity to mediate immune-dependent therapeutic effects. The second Lytix compound investigated, LTX-401, is an oncolytic amino acid derivative with potential immunogenic properties. We demonstrated that LTX-401 selectively destroys the structure of the Golgi apparatus. Subcellular fractionation followed by mass spectrometric detection revealed that LTX-401 was selectively enriched in the Golgi rather than in the mitochondria or in the cytosol. The Golgi-dissociating agent Brefeldin A (BFA) reduced cell killing by LTX-401 as it partially inhibited LTX-401-induced mitochondrial release of cytochrome c and the activation of BAX. The cytotoxic effect of LTX-401 was attenuated by the double knockout of BAX and BAK, as well as the mitophagy-enforced depletion of mitochondria, yet was refractory to caspase inhibition. LTX-401 induced all major hallmarks of immunogenic cell death. Moreover, LTX-401-treated tumors manifested a strong lymphoid infiltration. Altogether, these results support the contention that LTX-401 can stimulate immunogenic cell death through a pathway in which Golgi-localized LTX-401 operates upstream of mitochondrial membrane permeabilization

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4

Goere, Diane. "Caractérisation de la mort cellulaire induite par un anticorps trifonctionnel". Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T022/document.

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Le développement d’un cancer chez un individu immunocompétent témoigne, en partie, d’un échappement tumoral au système d’immunosurveillance. Par conséquent, la restauration ou l’induction de ces mécanismes de défense anti-tumorale est une des stratégies thérapeutiques actuelles. Un des principes de l’immunothérapie est basé sur l’injection d’anticorps ayant pour cible la cellule tumorale ou les cellules effectrices de l’immunité. L’efficacité anti-tumorale de ces anticorps a été considérablement améliorée par une meilleure compréhension des modes d’action et des effets modulateurs de ces anticorps. Ainsi, afin d’optimiser l’action des effecteurs immunitaires sur les cellules tumorales, un anticorps bispécifique, trifonctionnel, le catumaxomab, capable de se lier à la molécule d'adhérence des cellules épithéliales (EpCAM) exprimée par les cellules tumorales et à l'antigène CD3 des lymphocytes T, a été développé, essentiellement en traitement intra-péritonéal des ascites néoplasiques réfractaires.L’objectif de cette étude était de déterminer les effets immunomodulateurs du catumaxomab sur des cellules néoplasiques exprimant EpCAM, à partir de deux modèles expérimentaux (allogénique et autologue), de rechercher une cytotoxicité induite par la catumaxomab, et de la caractériser, notamment en analysant la présence ou non de signaux de stress inducteurs d’une mort immunogène tels que l’exposition membranaire de la calréticuline par les cellules tumorales pré-apoptotiques, la libération d’HMGB1 et d’adénosine triphosphate (ATP) dans le milieu extra-cellulaire, responsables d’une activation des lymphocytes T.En présence de cellules EpCAM+, le catumaxomab entrainait une activation majeure des lymphocytes T (expression de CD69, CD107a, HLA-DR et PD1), stimulait une réponse inflammatoire de type Thelper 1(Th1), et provoquait la synthèse d’interféron-gamma par les lymphocytes T CD8. Le catumaxomab engageait le CD16 (FcR) des cellules monocytaires et NK. De plus, sur des modèles allogéniques, le catumaxomab, provoquait une mort cellulaire associée à la libération d’ATP et induisait une mort immunogène après pré-incubation dans de l’oxaliplatine.Par conséquent, le catumaxomab permet de moduler l’environnement immunitaire dans les ascites néoplasiques, et de convertir une inflammation chronique et immunosuppressive (Th2) en une inflammation aigüe et immunogène (Th1). En revanche, dans ces conditions, l’administration seule de catumaxomab ne semble pas déclencher de mort immunogène.Différents moyens pourraient permettre d’améliorer la cytotoxicité de cet anticorps bispécifique : (1) le combiner avec un agent anti-néoplasique tel que l’oxaliplatine afin de promouvoir une mort immunogène, (2) affiner son action sur le CD3 des lymphocytes en modifiant sa configuration spatiale (anticorps BiTE), (3) amplifier son affinité pour le récepteur Fcdes cellules accessoires (Fc défucosylé), (4) augmenter sa cytotoxicité en modifiant la cible dirigée contre la molécule du système immunitaire (anti-PD-1…). Enfin, l’utilisation clinique pourrait être facilitée en humanisant cet anticorps chimérique murin afin d’éviter la formation d’anticorps anti-murins, dirigés contre le catumaxomab.Un essai thérapeutique de phase II dont le but est d’évaluer l’efficacité du catumaxomab intrapéritonéal après chirurgie de cytoréduction complète d’une carcinose gastrique, chez des patients ayant reçu en préopératoire une chimiothérapie systémique à base d’oxaliplatine vient de débuter. Au cours de cette étude, nous allons valider la capacité du catumaxomab 1) à induire un stress cellulaire immunogène et la mort des cellules cancéreuses, 2) à modifier la polarisation des cellules effectrices vers une maladie inflammatoire Th1, 3) à promouvoir l'expression des molécules de costimulation et TRAIL sur les cellules NK et monocytes, et corréler ces biomarqueurs immunitaires à l’efficacité du traitement
The development of cancer in an immunocompetent individual reflects, in part, a tumor escape from the immunosurveillance. The tumor escape is a complex, multifactorial, in which tumor cells will evade the defense mechanisms of the host by changing their microenvironment. Therefore, restoration or induction of these defense mechanisms is one of the therapeutic strategies against cancer. One of the principles of immunotherapy is based on the injection of antibodies that target tumor cells or effector cells of immunity. The anti-tumor efficacy of these antibodies has been greatly improved by a better understanding of modes of action and modulatory effects of these antibodies.Thus, to optimize the action of immune effectors to tumor cells, a bispecific antibody, trifunctional: catumaxomab, capable of binding to the adhesion molecule of the epithelial cells (EpCAM), expressed by tumor cells and the CD3 antigen of T cells, has been developed mainly in intraperitoneal treatment of refractory malignant ascites.The objective of this study was to determine the immunomodulatory effects of catumaxomab on tumoral cells expressing EpCAM, from two experimental models (allogeneic and autologous), evaluate and characterize cytotoxicity induced by catumaxomab, and analyze the presence of stress signals inducing immunogenic cell death such as membrane exposure of calreticulin by pre-apoptotic tumor cells, release of HMGB1 and of adenosine triphosphate (ATP) in the extracellular medium, inducing a T cell activation.In the presence of EpCAM + cells, catumaxomab induced a major the activation of T cells (expression of CD69, CD107a, HLA-DR and PD1), stimulated an inflammatory response Thelper type 1 (Th1) and the synthesis of interferon-gamma by CD8 T cells. Catumaxomab committed CD16 NK cells and monocytes. More, in models allogeneic catumaxomab, caused cell death associated with ATP release and induced an immunogenic cell death after pre-incubation of oxaliplatin.Therefore, catumaxomab modulates the immune environment in malignant ascites, and convert chronic and immunosuppressive inflammation (Th2) in acute and immunogenic inflammation (Th1). However, in these conditions, catumaxomab alone does not seem to trigger immunogenic cell death.the cytotoxicity of this bispecific antibody could be enhance by different techniques: (1) combining with chemotherapy such as oxaliplatin to promote immunogenic cell death, (2) refining its action on CD3 lymphocytes by changing its spatial configuration (BiTE antibody), (3) increasing its affinity for the FcR of accessory cells (Fc aglycosylated), (4) increasing its cytotoxicity by changing the target directed against the immune molecule (anti-PD-1 ...). Finally, the clinical use could be facilitated by this humanizing murine chimeric antibody to prevent the formation of anti-murine antibodies directed against catumaxomab.A phase II clinical trial aimed to evaluate the efficacy of intraperitoneal catumaxomab after complete cytoreductive surgery of gastric carcinomatosis in patients who received preoperative systemic chemotherapy with oxaliplatin have just started. In this study, we will validate the ability of catumaxomab 1) to induce immunogenic cell stress and death of cancer cells, 2) to change the polarization of effector cells to Th1 inflammatory disease, 3) to promote the expression of costimulatory molecules and TRAIL on NK cells and monocytes, and we will correlate these immune biomarkers to treatment efficacy

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5

Schlemmer, Frédéric. "Mécanismes de la chimiothérapie immunogène". Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T075.

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L’amélioration constante du pronostic des pathologies cancéreuses est le fruit des progrès réalisés dans leur prévention, leur dépistage, leur diagnostic et leur traitement. Malgré l’avènement récent des thérapies ciblées, la chimiothérapie conventionnelle reste souvent le seul recours pour des patients atteints de cancer non opérable ou non éligibles pour ces thérapeutiques novatrices. Certaines chimiothérapies conventionnelles (anthracyclines et oxaliplatine notamment) ont la capacité d’entrainer une mort des cellules tumorales dont les caractéristiques permettent d’induire une réponse immunitaire antitumorale efficace. Cette réponse immunitaire antitumorale spécifique agirait en synergie avec l’effet cytotoxique direct de ces drogues, participant ainsi à leur efficacité. La réponse immunitaire antitumorale induite par la chimiothérapie dépend de plusieurs mécanismes moléculaires et cellulaires clefs identifiés récemment. L’induction d’un stress du réticulum endoplasmique (RE) est à l’origine de l’exposition d’une protéine chaperonne résidente du RE, la calréticuline (CRT), à la surface des cellules mourantes, servant alors de signal de phagocytose pour les cellules dendritiques. La libération dans le milieu extracellulaire de signaux de danger est également essentielle : la protéine nucléaire High Mobility Group Box 1 (HMGB1) sert ainsi de ligand pour le Toll-like récepteur 4 (TLR4), présent à la surface des cellules dendritiques et son activation favorise l’apprêtement et la présentation des antigènes tumoraux aux lymphocytes T cytotoxiques. L’adénosine-5'-triphosphate (ATP) est également libéré par les cellules tumorales, entrainant l’activation des récepteurs purinergiques P2RX7 présents à la surface des cellules dendritiques, activant l’inflammasome NLRP3 et entrainant la libération d’IL-1 par les cellules dendritiques, favorisant alors l’orientation de la réponse immunitaire vers une réponse de type TH1 et la production d’interféron  par les lymphocytes T cytotoxiques. Dans ce travail, nous avons cherché à comparer la capacité de deux drogues issues d’une même classe de chimiothérapie, les sels de platine, à induire une mort cellulaire immunogène des cellules tumorales. Grace à des expériences in vitro et in vivo (modèles murins de vaccination antitumorale et de chimiothérapie sur tumeurs établies), nous avons pu montrer que l’oxaliplatine (OXP), contrairement au cisplatine (CDDP), avait la capacité d’induire une mort immunogène des cellules de cancer colique et que cette différence intra-classe dépendait de la capacité respective de ces deux drogues à entrainer un des phénomènes clés de l’induction d’une mort cellulaire immunogène, l’exposition de la CRT à la surface des cellules tumorales mourantes. Nous avons également pu montrer que l’induction d’une mort cellulaire immunogène des cellules de cancer colique par l’oxaliplatine avait une relevance clinique chez l’homme, l’existence d’un polymorphisme perte-de-fonction du gène tlr4 affectant le pronostic (survie sans progression) de patients traités par chimiothérapie pour un cancer colique métastatique. Par la suite, nous avons mis au point des biosondes permettant d’étudier à grande échelle, à l’aide d’une plateforme de vidéo-microscopie automatisée, la capacité de différentes drogues à induire les différents phénomènes clefs de la mort cellulaire immunogène des cellules tumorales (exposition de la CRT, libération d’HMGB1 et d’ATP). Nous avons ainsi pu montrer que la correction pharmaceutique du défaut d’activation d’un stress du réticulum endoplasmique par le cisplatine permettait de restaurer l’immunogénicité de la mort cellulaire induite par cette chimiothérapie. Ces résultats ouvrent la voie à la découverte de nouvelles molécules susceptibles, à elles seules ou en association à d’autres thérapies connues, d’améliorer le pronostic des néoplasies
The steady improvement of cancer prognosis is the result of progress in cancer prevention, screening, diagnosis and treatment. Despite the recent advent of targeted therapies, conventional chemotherapy often remains the only solution for patients with non-operable cancer or not eligible for these novel therapies.Some conventional chemotherapy (including anthracyclines and oxaliplatin) has the ability to cause tumor cells death with characteristics able to induce an effective antitumor immune response. This specific antitumor immune response would be synergistic with the direct cytotoxic effect of these drugs and contribute to their efficacy. The antitumor immune response induced by chemotherapy depends on several key cellular and molecular mechanisms recently identified. The induction of an endoplasmic reticulum (ER) stress is necessary for the exposure of calreticulin (CRT), an ER-resident chaperone protein, on the surface of dying cells, then acting as a phagocytosis signal for dendritic cells. Release of danger signals into the extracellular medium is also essential. The nuclear protein High Mobility Group Box 1 (HMGB1) is a ligand of the Toll-like receptor 4 (TLR4) on the surface of dendritic cells. TLR4 activation promotes the processing of tumor antigens and their presentation to cytotoxic T lymphocytes. Adenosine-5'-triphosphate (ATP) is also released by tumor cells, leading to the activation of the purinergic receptors P2RX7 expressed on the surface of dendritic cells, activating the NLRP3 inflammasome and causing the release of IL-1β by dendritic cells, while promoting the orientation of the immune response towards a TH1 response and the production of γ-interferon by cytotoxic T lymphocytes.In this work, we aimed to compare the ability of two drugs of a same class of chemotherapy, the platinum derivates oxaliplatin (OXP) and cisplatin (CDDP), to induce immunogenic death of tumor cells. Thanks to in vitro and in vivo experiments (models of tumor vaccination and chemotherapy on established tumors in mice), we showed that OXP, in contrast to CDDP, has the ability to induce immunogenic death of colon cancer cells. This intra-class difference depends on the ability of each drug to cause one of the key phenomena of immunogenic cell death: the induction of the exposure of the CRT to the surface of dying tumor cells. We could also show that the induction of immunogenic death of colon cancer cells by OXP had clinical relevance in humans. Indeed, the existence of a loss-of-function polymorphism of tlr4 affects the prognosis (PFS) of patients treated with OXP-based chemotherapy regimen for a metastatic colorectal cancer. Subsequently, we developed biosensors to study the ability of different drugs to induce key phenomena of cell death immunogen tumor cells (CRT exposure, HMGB1 and ATP release) using high-content screening by an automated video-microscopy platform. We showed that a pharmaceutical correction of the inability of cisplatin to induce an endoplasmic reticulum stress could restore the immunogenicity of cisplatin-induced tumor cell death. These results open the way to the discovery of new molecules that, alone or in combination with other known therapies, could improve the prognosis of cancer

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6

Ko, Adrien. "Impact de l’autophagie sur la radiosensibilité tumorale". Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T074.

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Les données existantes sur le rôle de l’autophagie dans la mort cellulaire radio-induite sont controversées et proviennent d’études pour lesquelles sont utilisées des drogues : l’action se produit donc de manière indirecte. Certains suggèrent que l’induction combinée de l’apoptose et de l’autophagie améliore le traitement par radiations ionisantes. D’autres indiquent que l’induction de l’autophagie favoriserait la radiorésistance des cellules tumorales et que l’utilisation d’inhibiteurs de l’autophagie augmenterait la réponse des tumeurs aux radiations ionisantes. L'autophagie, ou «self-eating», est un processus cellulaire activé par diverses conditions de stress, par lequel les cellules peuvent dégrader les protéines et les organites. Nous avons au cours de cette étude cherché à déterminer le rôle de l'autophagie dans la mort cellulaire radio-induite. Selon nos observations, l'autophagie est nécessaire pour la libération de l'ATP après un traitement par radiothérapie: en effet, le knockdown de gènes essentiels à l'autophagie limite la sécrétion d'ATP. Nous avons également constaté que des cellules déficientes pour l'autophagie traitées par radiothérapie sont incapables d’immuniser des souris contre une injection de cellules vivantes. En outre, les tumeurs déficientes pour l’autophagie répondent moins bien à un traitement par radiations ionisantes dans des souris immunocompétentes et continuent à proliférer, contrairement aux tumeurs “wild-type”. De plus, nous avons montré que les cellules déficientes pour l'autophagie ne sont pas en mesure de recruter des cellules dendritiques dans le lit tumoral. A l'inverse, l'inhibition des enzymes de dégradation de l’ATP extracellulaire accroît les concentrations d'ATP dans les tumeurs déficientes pour l'autophagie, ce qui rétablit le recrutement des cellules immunitaires dans le lit tumoral et restaure la réponse à la radiothérapie des cancers déficients pour l'autophagie. Ainsi, cette étude a montré l'importance de l'autophagie dans la réponse anti-tumorale spécifique, après traitement par radiations ionisantes. Ces résultats ouvrent de nouvelles perspectives pour comprendre la mort cellulaire radio-induite. Il reste cependant à découvrir les mécanismes moléculaires sous-jacents pour développer de nouvelles thérapies ciblées qui amélioreront l’efficacité de la radiothérapie
Most of the available data on autophagy and tumor response to IR comes from indirect conclusions after concomitant drug-IR exposure. Some authors suggest that concurrent induction of apoptosis and autophagy enhances radiation therapy. Oppositely, others indicate that the induction of autophagy contributes to the radioresistance of tumor cells and suggest that autophagy inhibitors may be employed to increase the sensitivity radioresistant tumors cells to ionizing radiation. Autophagy literally ‘self-eating’ is a cellular process activated in response to various conditions of cellular stress, whereby cells can liberate energy resources via the degradation of proteins and organelles. In this project we aimed to determine the potential role of autophagy in IR –induced cell death. We found that autophagy is required for the release of ATP in response to radiotherapy, as we observed that the knockdown of essential autophagy-related genes abolished its secretion. Furthermore, autophagy deficient tumors growing on immunocompetent mice did not respond to radiotherapy and continued proliferating in contrast to autophagy proficient tumors. We showed that autophagy deficient cells were neither able to recruit DCs into the tumor bed. Conversely, the inhibition of extracellular ATP degrading enzymes increased extracellular ATP concentrations in autophagy deficient tumors, which reestablished the recruitment of immune cells into the tumor bed, and restored radiotherapeutic responses in autophagy-deficient cancers.Altogether, this study showed the importance of autophagy in tumor-specific immune response after radiotherapy. Thus giving new insights into the concept of IR-induced cell death. However, there is still much that is unknown about molecular mechanisms that undergo IR-induced cell death. Understand these molecular mechanisms will help to develop new targeted therapies that will improve the effectiveness of radiotherapy

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7

Parney, Ian Frederick. "Human glioma immunology and immunogene therapy". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape8/PQDD_0003/NQ39580.pdf.

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8

Kjerrström, Zuber Anne. "Enhancement of HIV-1 DNA immunogens /". Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-304-x.

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9

Ljungberg, Karl. "Variable viral genes as genetic immunogens /". Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-399-6/.

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10

Karras, Marianna. "Evaluation of immunogen delivery by insects". Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409270.

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11

Humeau, Juliette. "Inhibition of Transcription by Dactinomycin Reveals a New Characteristic of Immunogenic Cell Stress". Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS492.

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La chimiothérapie constitue encore le traitement de référence pour la majorité des cancers. Or certains agents chimiothérapeutiques sont capables de déclencher des signaux de stress pre-mortem permettant d’activer une réponse immunitaire antitumorale et confèrent ainsi une protection à long terme. A l'aide d'un modèle construit par intelligence artificielle, nous avons identifié, parmi une librairie comprenant 50 000 composés, des agents anti-cancéreux qui, d'après leurs propriétés physico-chimiques, pourraient induire une mort cellulaire immunogène (ICD, de l'anglais "immunogenic cell death"). Cet algorithme nous a permis d'identifier la dactinomycine, qui, en effet, active les mécanismes sous-jacents à l'activation des cellules dendritiques in vitro et a un effet anti-cancéreux dépendant du système immunitaire in vivo. La dactinomycine, utilisée en clinique pour le traitement de sarcomes pédiatriques, est connue pour sa capacité à inhiber la transcription. Nous nous sommes donc demandé si d'autres inducteurs de l'ICD partageaient cette propriété. Différentes chimiothérapies immunogènes induisent en effet une inhibition de la synthèse d’ARN, qui est suivie d'une inhibition de la traduction et s’accompagne de l’activation des différentes voies de l’ICD. De plus, une étude rétrospective in silico révèle que les agents classés comme inhibiteurs de la synthèse d’ARN ou de protéines sont prédits comme étant immunogènes. Ces résultats montrent que l’inhibition de la transcription est un évènement précurseur essentiel à l’activation d’une mort cellulaire immunogène
Chemotherapy still constitutes the standard treatment for most cancers. Yet, some chemotherapeutics are able to trigger pre-mortem stress signals which activate an antitumor immune response and thereby confer long term protection. We used an established model built on artificial intelligence to identify, among a library of 50,000 compounds, anticancer agents that, based on their physicochemical characteristics, were predicted to induce immunogenic cell death (ICD). This algorithm led us to the identification of dactinomycin, which indeed activates the mechanisms preceding dendritic cell activation in vitro and demonstrates immune-dependent anticancer effects in vivo. Dactinomycin, mainly used to treat pediatric sarcomas, is known as able to inhibit transcription. We therefore investigated whether other ICD inducers would share this characteristic. Different immunogenic chemotherapeutics indeed inhibited RNA synthesis and secondarily translation, accompanied by an activation of ICD-related signaling. A retrospective in silico study revealed that agents annotated as inhibitors of RNA or protein synthesis are predicted as immunogenic. These results establish the inhibition of RNA synthesis as a major initial event for ICD induction

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12

Lundberg, Karin. "Arthritogenic and immunogenic properties of modified autoantigens /". Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-303-5/.

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13

Meanger, Jayesh. "Antigenic and immunogenic characterisation of avian reoviruses". Thesis, Meanger, Jayesh (1989) Antigenic and immunogenic characterisation of avian reoviruses. PhD thesis, Murdoch University, 1989. https://researchrepository.murdoch.edu.au/id/eprint/53041/.

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The antigenic relationship of 9 avian reoviruses of Australian origin was investigated by comparing the protein migration patterns of immunoprecipitated isotopically-labelled infected cell lysates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Close agreement with previously reported neutralisation test results and the precipitation of the σC protein was obtained. The results suggested that the σC protein was the major neutralisation protein, that it confers type-specificity to avian reoviruses, and is analogous to the g σ1 protein of mammalian reoviruses. Nine selected avian reovirus isolates were assigned to 4 antigenic groups based upon precipitation of the σC protein. Reassortants were produced between the parent viruses with the ability to replicate in Vero cells and those that were unable to replicate in Vero cells. Of the 9 reassortants examined, 8 were able to grow in Vero cells. Two gene segments, M2 and SI always originated from the parent virus (RAM-1) able to grow in Vero cells and never from the parent virus unable to grow in Vero cells. These results suggested that either the M2 or the SI gene segments were associated with the ability of the reassortant viruses to replicate in Vero cells, although 4 other gene segments in all the parent reoviruses used exhibited a similar migration rate during SDS-PAGE and their possible role in the replication of virus in Vero cells could not be determined. Temperature-sensitive (ts) mutants of the Vero cell-adapted RAM-1 strain of avian reovirus were induced for use as potential vaccine strains in Australia. During these experiments it was also determined that the parent RAM-1 strain already possessed ts characteristics. The virulence of one of the ts mutant (P20) selected for further study was compared to the parent virus and 2 other strains of avian reovirus. It was determined that both the parent RAM-1 virus and the ts mutant P20 had lower virulence for chickens than the 2 other strains of reovirus examined (724 and 1091) and that the virulence was dose-dependent. Multiple immunisation of chickens with the RAM-1 strain of virus resulted in the production of high titres of neutralising antibody to the homologous virus and to 2 other antigenically heterologous viruses (724 and 1091) with the passive transfer of these antibodies to progeny chickens via the egg yolk. Challenge experiments using high titred virus to induce tenosynovitis lesions showed that progeny chickens from hens immunised with the RAM-1 strain were protected against challenge with the homologous RAM-1 strain, were partially protected against challenge with the heterologous virus strain 724 but were not protected against challenge with the virus strain 1091. The results indicated that protection against avian reovirus infection in Australia would probably require the use of a polyvalent vaccine, and that selection of the appropriate vaccine strain should be based upon identification of the type-specific σC protein in isolates associated with disease.

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14

Chen, HwuDauRw 1958. "Growth of immunogenic skin tumors: Infiltrating leukocytes". Thesis, The University of Arizona, 1989. http://hdl.handle.net/10150/291654.

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Subpopulations of tumor infiltrating leukocytes in immunogenic skin tumors were identified with monoclonal antibodies. The tumors studied included primary UV-induced tumors and JB/MS melanomas, which survive in the host by immunosuppression of the immune response. The proportions of nucleated cells in primary UV-induced tumor cell suspensions which reacted with monoclonal antibodies were: 52% Mac-1+, 21% Lyt-1+, 13% Lyt-2+, 7% L3T4+, and 8% IL-2R+. Thus there was a high proportion of cells of the macrophage lineage in the growing UV-induced tumors. In JB/MS melanoma cell suspensions the mean proportion of macrophages was 6.4%, and total T lymphocytes (Lyt-1) averaged only 5.5%. Thus, there was little leukocytes infiltration into JB/MS melanoma, suggesting that chemotaxis was defective. The high level of macrophages and T cells in the primary UV-induced tumors indicates that chemotaxis was intact. Therefore, either the tumorcidal capacities of the macrophages and Tc were insensitive to activated macrophages and to Tc cells.

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15

Zheng, Biao. "Inductive interactions between antigen presenting cells and T cells". Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314796.

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16

Coral, Didem. "Studies On Novel Immunogenic Proteins Of Clostridium Chauvoei". Master's thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/12611360/index.pdf.

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Clostridium chauvoei is a gram-positive, spore-forming anaerobic bacterium. It is the pathogenic agent of blackleg, a disease causing serious toxemia and high mortality in cattle, sheep and many other domestic and wild animals. It is considered the most important Clostridium producing economic losses in livestock. Typically, animals infected with blackleg die rapidly without any signs of illness. Animals quickly die within 12 to 48 hours after contracting the disease. Therefore, the control of this disease is done by commercial vaccines consisting of whole formolized cultures. Immunity against C. chauvoei is associated with whole cell, including its somatic and flagellar antigens while in other clostridial diseases, protective immunity is obtained by the use of vaccines containing toxoids. Moreover, it is essential to obtain new information about the somatic antigens of C. chauvoei. Proteomics is the study of the proteome, the protein complement of the genome. The proteome has been defined as the entire complement of proteins expressed by a cell, organism, or tissue type, and accordingly, proteomics is the study of this complement expressed at a given time or under certain environmental conditions. 2-DE with Immobilized pH Gradients (IPGs) combined with protein identification by Mass Spectrometry (MS) is currently the workhorse for proteomics. Much of information about immunogenic component can be derived from proteomics coupled to Western blotting, namely immunoproteomics. Our study constitutes the first immunoproteomic analysis of C. chauvoei to identify candidate immunogenic antigens for development of new vaccines. Analyses were performed by Western blot and dot blot techniques against the whole cell extract proteins of C. chauvoei separated by 2-DE. Firstly, the growth conditions of two different strains, C. chauvoei ATCC 11957 and C. chauvoei 20 were optimized. After mice immunization studies with experimental vaccines prepared, sera were obtained for evaluation of the immunoglobulin G antibody level by ELISA. After high level of antibody response determination, 1-DE, 2-DE and immunoblot studies were performed for the characterization of immunogenic proteins. In the study, a total of 460 protein spots could be detected on the 2-DE gels by the help of Delta2D image analysis software and 30 of them were reacted with polyclonal antibodies against inactivated whole cells of C. chauvoei. Among these 30 spots, and 8 of them could be characterized by MALDI-TOF MS analyses. Of these 8 spots revealed four different gene products (distinct ORFs). Ornithine decarboxylase, methionine adenosyltransferase, glucose-6-phosphate isomerase, and flagellin protein FliB (C) are the characterized proteins. Glucose-6-phosphate isomerase has been identified as an immunogenic protein for a pathogenic microbe and in C. chauvoei for the first time. Methionine adenosyltransferase and ornithine decarboxylation were identified as immunogenic for C. chauvoei for the first time. The last defined protein is the flagellin protein FliB(C) which is known to be major immunogenic protein of C. chauvoei.

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17

Loskog, Angelica. "Immunogene Therapy of Bladder Carcinoma : A Preclinical Study". Doctoral thesis, Uppsala universitet, Enheten för onkologi, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-2637.

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This thesis comprises studies on murine and human models of bladder carcinoma with the aim to develop novel immunogene therapies. On the basis of the results presented in this thesis, a clinical trial is underway. The potential of activating the immune system to combat cancer has long intrigued immunologists. Research has now been intensified and clinically effective treatments are beginning to materialize. We evaluated the induction of anti-tumor responses by inserting immunomodulating genes into tumor cells with adenovectors. Human biopsies and cell lines were positive for adenovirus attachment receptors, and cell lines were easily transduced. CD40L modified cells efficiently induced maturation of dendritic cell (DC). Phenotypical changes of AdCD40L transduced cells, such as increased apoptotic rate, upregulated MHC-I, Fas and TNFR may further strengthen the anti-tumor response. CD40L modified murine bladder cancer cells activated systemic immunity upon vaccination and in situ injections of AdCD40L inhibited tumor progression. Cytotoxic assays revealed the presence of cytotoxic T cells (CTLs) in vaccinated mice. Many tumors have developed ways to evade the immune system. Bladder carcinoma is associated with immune escape mechanisms like IL10 production. We demonstrated that immunosuppression by IL10 inhibited CTL function and that IL10 suppression may be reverted by AdCD40L therapy. In conclusion, AdCD40L therapy induces systemic immunity and inhibits tumor progression in murine models. The immunological mechanisms involve maturation of nearby DCs and CTL induction. AdCD40L therapy is effective despite immune escape mechanisms, e.g. IL10 secretion. The thesis argues for using AdCD40L immunogene therapy as a treatment of bladder carcinoma.

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18

Martin, Spencer David. "Identifying and targeting immunogenic mutations in ovarian cancer". Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/58279.

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A hallmark of cancer is the accumulation of mutations, and a small proportion of these give rise to mutant neoantigens – mutated peptides bound to Major Histocompatibility Complex (MHC) and recognized by T cells. Accumulating evidence suggests that mutant neoantigens (hereafter referred to as “neoantigens”) underlie successful immune therapies in cancers with high mutation loads, such as melanoma. Moreover, neoantigen-specific vaccines have successfully targeted highly mutated murine tumor models. However, less is known about neoantigen-specific T cell responses in cancers with moderate mutation loads, such as ovarian cancer. I hypothesized that (1) modified peptide-based vaccination schedules can lead to enhanced antigen-specific T cell responses; (2) neoantigen-specific vaccines can elicit T cell responses that eradicate murine ovarian tumors; and (3) neoantigen-reactive T cells are detectable in human ovarian tumors and peripheral blood. To activate high frequencies of antigen-specific T cells, I developed a vaccination method involving repeated, daily immunizations with long peptides and adjuvant. This method elicited robust T cell responses that eliminated established murine tumors. I used these enhanced vaccination methods to target tumor-specific mutations identified by exome- and RNA-sequencing of the ovarian tumor model ID8-G7. Prophylactic and therapeutic vaccinations were performed targeting all expressed mutations that had a predicted MHCI binding affinity < 1500 nM (n=17 mutations). Though the vaccines elicited robust T cell responses to most mutations, activated T cells failed to recognize ID8-G7 tumors in vitro and failed to control tumor growth in vivo, indicating that none of the evaluated mutations represented authentic neoantigens. I also investigated the neoantigen-specific T cell repertoire in blood and tumor samples donated by an ovarian cancer patient. A neoantigen-specific T cell response was identified in the first recurrence tumor sample, and a derived T cell clone recognized tumor from each of three time points. Furthermore, peripheral blood samples donated prior disease recurrence harbored T cells recognizing the same mutation. The results presented here show that neoantigen-specific T cells can arise spontaneously in ovarian tumors and are detectable in peripheral blood; however, low mutation burdens in ovarian cancer may limit the utility of neoantigen-targeted vaccines to a minority of patients with this disease.
Medicine, Faculty of
Graduate

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19

Carlring-Wright, Jennifer. "Immunogene therapy for the treatment of uveal melanoma". Thesis, University of Sheffield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392929.

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20

Donaldson, Matthew. "Antimicrobial carbohydrate vaccines : development of Burkholderia pseudomallei immunogens". Thesis, University of East Anglia, 2013. https://ueaeprints.uea.ac.uk/48103/.

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The potential bio-terror threat posed by Burkholderia pseudomallei highlights the need for an effective vaccine. Immunisation and challenge experiments in mice have demonstrated that the capsular polysaccharide (CPS-1) of B. pseudomallei, which is composed of β-1,3-linked 6-deoxy-D-manno-heptopyranose residues, is a promising candidate for vaccine development. This thesis set out to explore routes to potential vaccine candidates for Burkholderia pseudomallei infection based on both bottom-up synthetic monosaccharide constructs and top-down CPS-1 conjugates. Initially this thesis focused on the development of a monosaccharide antigen based on the 6-deoxy-D-manno-heptose building block of CPS-1. The antigens were conjugated to TetHc carrier protein via an 8-(methoxycarbonyl)octyl linker using traditional acyl hydrazide conjugation chemistry. The TetHc monosaccharide conjugates were analysed by SDS-PAGE and MALDI-ToF mass spectrometry before undergoing immunisation trials in sheep. Immunological assessment of the resulting polyclonal antisera was conducted by ELISA, slot-blot and the Octet biosensor system. These studies demonstrated the generation of antibodies that were specific for the cognate monosaccharide. Preliminary investigations showed that the 6-deoxy-D-mannoheptose- derived antibodies reacted positively with the natural CPS-1. The linker was shown to be important in the specificity of the polyclonal sera, leading to cross reactivity with non-parent monosaccharide antigens. A second generation of antigens was developed using a short linker, 3-(methyl mercaptopropionate). The expression and purification of the natural B. pseudomallei CPS-1 was achieved in an avirulent strain of Burkholderia, B. thailandensis E555. Characterisation of the capsular polysaccharide highlighted the co-expression of an α-1,3-mannan polysaccharide. Mild acid hydrolysis of the CPS-1 preparation, combined with capillary electrophoresis was trialled with a view to the generation of oligosaccharide fragments of CPS-1 for conjugation and immunisation studies. While initial carbohydrate conjugates were prepared with a previously described tetanus toxoid Hc fragment, the development and in planta expression of a chemically conjugatable Hepatitis B core virus-like particle system was also achieved.

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21

DELMOTTE, CAROLINE. "Etude physicochimique et internalisation cellulaire d'un peptide immunogene". Orléans, 1999. http://www.theses.fr/1999ORLE2032.

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Dans le cadre du developpement de vaccins sous-unites, l'immunogenicite de peptides synthetiques contenant un epitope t cytotoxique (np6) et un epitope t auxiliaire (t) issus de proteines du virus de la rougeole a ete etudiee. Le peptide chimere tt-np6 a pour interet d'induire une reponse t cytotoxique en absence d'adjuvants et de composes hydrophobes. Le mecanisme de reconnaissance d'un antigene par les lymphocytes t ainsi que les nouvelles approches vaccinales capables d'induire une reponse immune cellulaire sont decrits succinctement. Pour stimuler in vivo des lymphocytes t cytotoxiques (ctl), l'epitope t cytotoxique doit etre internalise dans le cytosol. C'est pourquoi nous etudions l'internalisation cellulaire et l'affinite pour la membrane plasmique des peptides tt-np6, t-np6 et tt. Les peptides marques par l'oregon green 514 ont ete localises par microspectrofluorimetrie confocale au niveau de la membrane cellulaire. Le peptide le plus immunogene tt-np6 se differencie des peptides tt et t-np6 par une internalisation plus profonde dans le cytosol et une accumulation de type vesiculaire. L'internalisation est directement reliee a la presence du peptide a la membrane plasmique. Des etudes physicochimiques ont ensuite montre que le peptide tt-np6 a une plus grande affinite pour les lipides, qu'il est plus helicogene et plus stable a la proteolyse que t-np6 et tt. L'etude approfondie de la structure de tt-np6 a mis en evidence une propension a s'autoassocier, ce qui favoriserait la resistance a la proteolyse, et elle a permis d'identifier une arginine qui serait responsable d'une perte d'helicite et dont la mutation ameliorerait l'activite biologique du peptide. Enfin, le peptide tt est etudie du point de vue de son affinite pour des alleles humains et de son helicogenicite afin de permettre son application a d'autres epitopes. Ce travail souligne l'importance des analyses physicochimiques dans l'elaboration de vaccins sous-unites capables de stimuler des ctls.

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22

Pramanpol, Nuttawan. "Structural studies on immunogenic proteins of Burkholderia pseudomallei". Thesis, University of Sheffield, 2013. http://etheses.whiterose.ac.uk/3807/.

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McCormick, Adele. "Enhancement of the immunogenicity of recombinant gp120 of HIV-1". Thesis, University of Sheffield, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366115.

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24

Ali, Daham Hassan. "Studies on the antigenicity of citrate synthase". Thesis, University of Bath, 1989. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233608.

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Crisci, Elisa. "Immunogenic properties of calicivirus-like particles as vaccine vectors". Doctoral thesis, Universitat Autònoma de Barcelona, 2011. http://hdl.handle.net/10803/83962.

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Las nuevas vacunas de subunidades están abriéndose paso dentro de la vacunología veterinaria y entre ellas, las pseudopartículas virales o VLPs (por su nombre en inglés “virus-like particles”) son una de las estrategias más atractivas que están abriendo nuevas fronteras en la vacunación de animales. Las VLPs son estructuras proteicas rígidas con un tamaño dentro del rango de los nanómetros, que presentan una geometría muy bien definida y una espectacular uniformidad que mimetiza la estructura de los virus nativos de los que proceden. Las VLPs tienen importantes ventajas en relación a la seguridad por el hecho de carecer de genoma viral, que elimina cualquier riesgo asociado a la replicación viral, reversión, recombinación o reorganizamiento genómico. La proteína de la cápside del virus de la fiebre hemorrágica del conejo (por su nombre en inglés “Rabbit haemorrhagic disease virus” o RHDV) es capaz de formar RHDVVLPs y estas partículas han demostrado poseer una fuerte inmunogenicidad, protegiendo al hospedador natural tras un desafío mortal. Además, estudios anteriores apuntaron la posibilidad de usar RHDV-VLPs como vector para mejorar la inmunoterapia contra el cáncer. Sin embargo, no hay estudios que hayan investigado la posibilidad de usar las RHDV-VLPs como vector vacunal con epitopos de enfermedades virales de animales. El objetivo de esta tesis es estudiar el potencial inmunogénico de las RHDV-VLPs como vectores vacunales de enfermedades virales en diferentes animales. En los dos primeros estudios, se investigó la inmunogenicidad de las RHDV-VLPs en el modelo murino, tanto in vitro como in vivo. Los resultados de estos estudios demostraron que el epítopo insertado era procesado y presentado mediante MHC-I por células dendríticas y que la inmunogenicidad dependía de los diferentes sitios de inserción. Las RHDV-VLPs quiméricas fueron capaces de proteger a los ratones frente a un desafío viral. También, la respuesta se vio alterada según la ruta de administración del antígeno. El tercer estudio confirmó los resultados en ratón, pero esta vez en experimentos in vitro con células de cerdo. Por último, se estudió inmunogenicidad de las RHDV-VLPs quiméricas en cerdo. Los resultados mostraron que la ruta de administración y el adjuvante determinaron la respuesta inmune después de la inmunización con las RHDVVLPs quiméricas y que los animales presentaban muy buena respuesta inmune celular y humoral, no solo frente a RHDV-VLPs sino frente al epítopo antigénico. Estudios posteriores se tendrán que abordar para demostrar la protección de los cerdos. En conclusión, en esta tesis se demuestra el potencial de las RHDV-VLPs como inmunógenos en dos sistemas animales diferentes.
New subunit vaccines are getting a foothold in veterinary vaccinology and virus-like particles (VLPs) are one of the most appealing approaches opening up frontiers in animal vaccines. VLPs are robust protein cages in the nanometer range exhibiting welldefined geometry and remarkable uniformity that mimic the overall structure of the native virions. VLPs have an important advantage in terms of safety; indeed, lacking the genome of the virus avoid any of the risks associated with virus replication, reversion, recombination or re-assortment. Rabbit haemorrhagic disease virus (RHDV) capsid protein is able to form RHDV-VLPs and these particles showed a strong immunogenicity and protected the natural host after a lethal challenge. Additionally, previous studies described the possibility to use RHDV-VLPs as platform for the insertion of foreign epitopes or for DNA packaging. Nowadays, one study has shown the possibility to use RHDV-VLPs as carrier for improving cancer immunotherapies but no studies have investigated the possibility to use RHDV-VLPs as vaccine vectors carrying epitopes corresponding to viral animal diseases. This thesis is aimed to study the potential immunogenicity of RHDV-VLPs as epitope carriers for viral disease in different animal models. In the first two studies, the immunogenicity of chimeric RHDV-VLPs was investigated in a murine system in vitro and in vivo. Results from these studies demonstrated that the inserted epitope was processed and presented in an MHC-I context by dendritic cells (DCs) and that the different sites of insertion of the epitope influenced the immunogenicity of the VLPs. Chimeric RHDV-VLPs were able to protect mice from a viral challenge. Also, the route of antigen delivery influenced the immunogenicity of the particles. The third study confirmed the initial results but this time in in vitro experiments using porcine cells. Lastly, chimeric RHDV-VLPs were studied as immunogens in pigs. The results showed that the delivery route and adjuvant influenced immune responses after chimeric RHDV-VLP immunization and more importantly that pigs exhibited very good cellular and humoral immune responses against not only RHDV-VLPs but also against the antigenic epitope. Further studies have to be performed to prove protection in pigs. In conclusion, in this thesis we demonstrated the potential of RHDV-VLPs as immunogens in two different animal systems.

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26

Marguerie, Monique. "Combining the Immunogenic Cancer Mutanome with Oncolytic Virus Therapy". Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31409.

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Oncolytic viruses (OVs) are effective anti-cancer agents, however their abilities to induce anti-tumor immunity are not yet optimal. Mutanome epitopes are a novel source of tumor antigen formed as a result of mutations within the tumor genome. Within this project we attempted to combine B16F10 mutanome vaccination with OV therapy. We confirmed previous findings that significant immune responses to these epitopes can be generated. Furthermore, we designed and cloned a multi-epitope mutanome construct into MG1 Maraba virus and E1-/E3- deleted type 5 Adenovirus to use for heterologous prime-boost vaccination. While we demonstrated that these viruses induced T-cell responses to one mutanome epitope, we failed to detect responses to the other epitopes. Furthermore there was no effect seen on overall survival. This approach warrants further investigation because coupling mutanome vaccination with OV therapy has the potential to exploit the therapeutic effects of the OV while inducing anti-tumor immunity to tumor-unique antigens.

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27

Tedcastle, Alison. "The immunogenic epitopes of the glycoproteins of human metapneumovirus". Thesis, University of Newcastle Upon Tyne, 2010. http://hdl.handle.net/10443/1208.

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Recently discovered in 2001, human metapneumovirus (HMPV) is a member of the Paramyxoviridae family and is a major cause of respiratory tract infections in infants and young children as well as the elderly and immunocomprimised. In the related pneumovirus RSV, the two major surface glycoproteins, F and G are protective antigens in animal models although F is highly conserved and G highly variable. In this study, the equivalent glycoproteins of HMPV, F and G, were cloned into vaccinia virus to allow expression of the individual proteins. These recombinants were utilised for the generation of both monoclonal and glycoprotein specific polyclonal antibodies. Immunofluorescence studies revealed that the anti-F protein specific antibodies were cross reactive between both sub-groups and that anti-G antibodies were to a lesser extent, also cross reactive. These antibodies were also shown to neutralise homologous virus. Whilst anti-F protein antibodies also neutralised a heterologous strain of HMPV, so did anti-G antibodies directed towards a sub-group B but not a sub-group A strain. Western blotting with F and G glycoprotein specific anti-sera was unsuccessful due to high levels of non-specific reactivity in the sera. The generation of monoclonal antibodies towards the G glycoprotein was attempted by means of a novel screening system using inactivated recombinant vaccinia virus. However, due to the non-specific reactivity of the hybridomas with vaccinia virus, only one anti-G antibody was isolated along with two anti-F MAbs and one antibody directed towards an internal HMPV protein. Further characterisation of this antibody, by western blotting, indicated it was directed towards the phosphoprotein. The third surface glycoprotein of HMPV, SH, is larger than the equivalent in RSV and unlike the latter, may also play a role in protective immunity. Attempts to clone the HMPV SH gene identified several mutations in the sequence resulting in truncation of all or the majority of the lumenal domain of the proteins arising on adaption to replication in cell culture. SH glycoprotein specific antibodies generated against the recombinant vaccinia virus expressing the mutated SH protein of the B1 v strain of virus were cross reactive with an A2 strain in immunofluorescence studies and also neutralised wild type HMPV. The tendency of the virus to mutate on adaptation to replication in cell culture frustrated attempts to establish an animal model. In mice, whilst low passage virus, with a mixed population of wild type and mutant virus, resulted in a productive infection, high passage virus, with no functional SH glycoprotein produced an abortive infection with evidence of genome replication and transcription but no release of infectious virus. However, in challenge studies with mutant viruses mice immunised with HMPV were protected when challenged with the homologous strain, however, immunisation of mice with recombinant vaccinia virus expressing the G glycoprotein showed no signs of protection against challenge suggesting that in this animal model, the G glycoprotein is not a protective antigen.

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28

Setiyaningsih, Surachmi. "Molecular and immunogenic analysis of Jembrana disease virus Tat". Thesis, Setiyaningsih, Surachmi (2006) Molecular and immunogenic analysis of Jembrana disease virus Tat. PhD thesis, Murdoch University, 2006. https://researchrepository.murdoch.edu.au/id/eprint/299/.

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Jembrana disease is an acute and severe disease of Bali cattle (Bos javanicus) endemic in Indonesia that is caused by a bovine lentivirus designated Jembrana disease virus (JDV). Previous studies have demonstrated that it is possible to induce a protective immunity against the disease by immunisation with a crude whole virus vaccine prepared from the tissues of infected cattle. This vaccine has been demonstrated to ameliorate the clinical signs of disease resulting from exposure to virus infection but a safer vaccine amenable to commercial production techniques is required. JDV, like all lentiviruses, encodes a transcriptional trans-activator Tat protein that is encoded from one or both of two exons of the tat gene. Tat is particularly essential for virus replication and it was hypothesised that the induction of an immune response in cattle against JDV Tat may effect protection against virus infection. Investigations were therefore conducted on JDV Tat to provide basic information on the protein that would enable it to be further investigated as a potential immunogen for incorporation into vaccines for the control of Jembrana disease. Analysis of tat transcripts obtained from tissues of cattle infected with three strains of JDV suggested that, during the acute clinical disease, Tat produced at this stage of the disease process was translated from the first coding exon only. Nucleotide variation in this exon, which would have translated into amino acid variations in the Tat protein, was evident especially between strains from geographically different regions of Indonesia. There was; however, conservation of the essential functional domains of cysteine-rich, core and basic regions, which suggested immunity to a single Tat protein might protect against infection by heterologous strains. Subsequent studies on Tat reported in the thesis therefore concentrated on the protein encoded by tat exon 1 of a single strain of JDV. The exon 1 of tat was cloned into the pGEX vector and recombinant Tat expressed in Escherichia coli. Methods for the purification of the expressed protein were developed. Immunogenicity of the recombinant protein was initially demonstrated by inoculation of the protein into a sheep which developed a high titred specific antibody response. Antibodies induced by this recombinant protein recognised native Tat proteins produced by three JDV strains in Bali cattle and provided a valuable reagent for the subsequent detection of Tat in vitro and in vivo. Aspects of the antibody response to Tat were determined in cattle that had been infected naturally or experimentally with JDV, and compared with the levels of antibody to the immunodominant capsid protein. Tat antibodies were detected in 23 % of 128 Bali cattle from Jembrana disease-endemic areas of Indonesia; in all these cattle, evidence of previous virus infection had been demonstrated by detection of antibody to the JDV capsid protein by Western blot analysis. In cattle experimentally infected with JDV, low levels of serum antibody to Tat were detected by Western blot in the first month post-infection but the levels of antibody then decreased; levels of antibody to the JDV capsid protein increased over the 6-month observation period following infection. The detection of Tatantibody soon after the acute clinical disease suggested that this protein is secreted extracellularly during JDV infection in cattle. In contrast to the antibody response to Tat in JDV-infected cattle, an apparently greater antibody response to Tat was induced by injection of recombinant Tat in Bali cattle. The strong antibody response resulting from inoculation of the recombinant Tat and low levels of Tat antibody in animals that had been naturally or experimentally infected with virus suggested there might be a conformational difference in the recombinant and native Tat protein and that the native protein was a poor immunogen, or that the levels of Tat in infected cattle were too low to induce a strong antibody response. As an alternative means of inducing an immune response to JDV Tat, perhaps one associated with a greater cell-mediated rather than an antibody response, a candidate tat DNA vaccine was produced by insertion of tat exon 1 into a DNA vaccine vector. Transfection of this naked DNA plasmid into mammalian cells induced the expression of a functional Tat protein which maintained antigenicity. The results suggested this construct merits further animal studies attempting to induce a protective immune response against Jembrana disease in cattle. A method of assaying the trans-acting function of Tat was also developed which will have application for quality control procedures for large-scale production of tat DNA vaccine.

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29

Setiyaningsih, Surachmi. "Molecular and immunogenic analysis of Jembrana disease virus Tat". Setiyaningsih, Surachmi (2006) Molecular and immunogenic analysis of Jembrana disease virus Tat. PhD thesis, Murdoch University, 2006. http://researchrepository.murdoch.edu.au/299/.

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Jembrana disease is an acute and severe disease of Bali cattle (Bos javanicus) endemic in Indonesia that is caused by a bovine lentivirus designated Jembrana disease virus (JDV). Previous studies have demonstrated that it is possible to induce a protective immunity against the disease by immunisation with a crude whole virus vaccine prepared from the tissues of infected cattle. This vaccine has been demonstrated to ameliorate the clinical signs of disease resulting from exposure to virus infection but a safer vaccine amenable to commercial production techniques is required. JDV, like all lentiviruses, encodes a transcriptional trans-activator Tat protein that is encoded from one or both of two exons of the tat gene. Tat is particularly essential for virus replication and it was hypothesised that the induction of an immune response in cattle against JDV Tat may effect protection against virus infection. Investigations were therefore conducted on JDV Tat to provide basic information on the protein that would enable it to be further investigated as a potential immunogen for incorporation into vaccines for the control of Jembrana disease. Analysis of tat transcripts obtained from tissues of cattle infected with three strains of JDV suggested that, during the acute clinical disease, Tat produced at this stage of the disease process was translated from the first coding exon only. Nucleotide variation in this exon, which would have translated into amino acid variations in the Tat protein, was evident especially between strains from geographically different regions of Indonesia. There was; however, conservation of the essential functional domains of cysteine-rich, core and basic regions, which suggested immunity to a single Tat protein might protect against infection by heterologous strains. Subsequent studies on Tat reported in the thesis therefore concentrated on the protein encoded by tat exon 1 of a single strain of JDV. The exon 1 of tat was cloned into the pGEX vector and recombinant Tat expressed in Escherichia coli. Methods for the purification of the expressed protein were developed. Immunogenicity of the recombinant protein was initially demonstrated by inoculation of the protein into a sheep which developed a high titred specific antibody response. Antibodies induced by this recombinant protein recognised native Tat proteins produced by three JDV strains in Bali cattle and provided a valuable reagent for the subsequent detection of Tat in vitro and in vivo. Aspects of the antibody response to Tat were determined in cattle that had been infected naturally or experimentally with JDV, and compared with the levels of antibody to the immunodominant capsid protein. Tat antibodies were detected in 23 % of 128 Bali cattle from Jembrana disease-endemic areas of Indonesia; in all these cattle, evidence of previous virus infection had been demonstrated by detection of antibody to the JDV capsid protein by Western blot analysis. In cattle experimentally infected with JDV, low levels of serum antibody to Tat were detected by Western blot in the first month post-infection but the levels of antibody then decreased; levels of antibody to the JDV capsid protein increased over the 6-month observation period following infection. The detection of Tatantibody soon after the acute clinical disease suggested that this protein is secreted extracellularly during JDV infection in cattle. In contrast to the antibody response to Tat in JDV-infected cattle, an apparently greater antibody response to Tat was induced by injection of recombinant Tat in Bali cattle. The strong antibody response resulting from inoculation of the recombinant Tat and low levels of Tat antibody in animals that had been naturally or experimentally infected with virus suggested there might be a conformational difference in the recombinant and native Tat protein and that the native protein was a poor immunogen, or that the levels of Tat in infected cattle were too low to induce a strong antibody response. As an alternative means of inducing an immune response to JDV Tat, perhaps one associated with a greater cell-mediated rather than an antibody response, a candidate tat DNA vaccine was produced by insertion of tat exon 1 into a DNA vaccine vector. Transfection of this naked DNA plasmid into mammalian cells induced the expression of a functional Tat protein which maintained antigenicity. The results suggested this construct merits further animal studies attempting to induce a protective immune response against Jembrana disease in cattle. A method of assaying the trans-acting function of Tat was also developed which will have application for quality control procedures for large-scale production of tat DNA vaccine.

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30

Eger, Lars. "Immunogeneic Cell Populations of the Skin". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2008. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1213729820693-69540.

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Dendritic cells (DCs), a hematopoietic cell type belonging to the sub-group of cells called antigen presenting cells (APCs), inhabit a central role in innate and adaptive immunity. Although the DC family is very heterogeneous, all members share unique features. Most importantly, DCs can stimulate an immune response. This is due to the cells’ ability to capture and process antigens and to maturate in the presence of danger signals presented by pathogens. Maturation in turn results in the migration of DCs from the tissue they reside in to the draining lymph nodes, as well as in the subsequent presentation of the acquired antigens to T cells. In the skin, which is one of the most immunogeneic organs, DCs are present in sizable numbers in both the epidermis and the dermis. This study focused on two types of DCs: epidermal Langerhans cells (LCs) and dermal DCs (DDCs). While much is understood about LCs, far less is known about the role that DDCs play in skin immunity. Therefore one purpose of this study was to characterize DDCs and to compare their phenotype and functions to that of LCs. This study used two different methods to characterize human skin resident immune cells with regard to their number and distribution. First, a stable analytical immunohistochemistry-based method was developed and applied to a substantial number of healthy skin donors. This enabled a quantitative analysis of skin DC types and skin resident T cells at different anatomical locations in situ. A novel method to count dermal cell populations in situ was developed that resulted in the first published quantification of APCs, DDCs, as well as T cells in human dermis. Second, the traditional form of the emigration assay, which selectively enriches vital cells capable of ex vivo emigration from the skin, was upgraded toward a stable analytical method to separate epidermal LCs from DDCs. In this way, both skin DC types became accessible in sufficient numbers to allow for a comparison of phenotypes and functions in vitro. The resulting phenotypic observations clearly showed that both, LCs and DDCs are not fully mature after their emigration ex vivo and that both can be transformed into a phenotypically more mature state by treating them with inflammatory cytokines. What’s more, LCs are also functionally in an immature state after their emigration. They efficiently took up antigen, showed a low capacity to trans-migrate in response to chemokines, and demonstrated a low capacity to stimulate allogeneic T cells in a mixed leukocyte reaction (MLR). For the first time this study observed all these main APC functions not only for LCs but additionally for DDCs. As these observations were made in relation to LCs of the same donor, it could be concluded that DDCs are functionally more mature than LCs after emigration. DDCs showed a lower antigen uptake capacity than LCs but were superior in terms of their migratory and stimulatory capacity. However, treatment with cytokines could skew LC functions toward functional capacities observed for DDCs, i.e., it decreased LCs’ Ag uptake and increased their migratory and stimulatory capacity, whereas the cytokine treatment did not alter DDCs’ functional capacities. After improving immuno-histochemistry and the emigration assay using healthy skin samples, these newly developed techniques were implemented in clinical trials to observe the number, distribution and migratory capacity of skin DCs and T cells in patients undergoing allogeneic hematopoietic cell transplantation (aHSCT). Such a study is of importance because the turnover of DCs and T cells is closely associated with the occurrence of acute graft-versus-host disease (aGvHD), the major cause of morbidity and mortality after aHSCT. Due to the study design used, this study concisely demonstrate that at the onset of aGvHD, different DC types accumulate along with effector T cells in skin lesions of aGvHD but not in uninvolved skin of the same patient. These results suggest that in addition to donor T cells LCs and DDCs play a role during the early phase of cutaneous aGvHD directly within the site of inflammation. The view of many authors that DC depletion in the transplant recipient, especially in target organs, is a promising approach for aGvHD prophylaxis and therapy is further underscored by these results. One targeting strategy to inhibit GvHD by eliminating recipient DCs may be the use of DC specific monoclonal antibodies. Alemtuzumab (anti-CD52) is a monoclonal antibody and has proven effective in preventing aGvHD after aHSCT. It may, despite depleting donor T cells, also work by targeting recipient DCs. To determine whether the last mechanism of action is significant, a second clinical study investigated the effects of intravenous alemtuzumab on DCs by comparing the number of these cells in skin and blood of patients before and after a 4-week course of alemtuzumab treatment. The result was that although skin DCs weakly express the target antigen CD52 the number of these cells was not consistently reduced by alemtuzumab. In contrast, circulating blood DCs have a stronger CD52 expression and were significantly reduced by the treatment. In conclusion, this work provides new insights into the phenotypical and functional characteristics of human skin DCs, as well as into the fate of these cell types during aHSCT. The investigation of the APC system during aGvHD as carried out here will help to understand the process of aGvHD in more detail. All these efforts may hopefully support the development of new approaches for therapy and prevention of this major limitation of aHSCT and may help to improve this only curative therapy for several life-threatening diseases.

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31

Pons-Tostivint, Elvire. "Stratégies thérapeutiques innovantes pour stimuler la réponse immune antitumorale de cytotoxiques utilisés pour le traitement des cancers du sein". Thesis, Toulouse 3, 2021. http://www.theses.fr/2021TOU30216.

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Cette dernière décennie, de nombreuses données cliniques et pré-cliniques ont démontrées le rôle primordial du système immunitaire dans l'efficacité des chimiothérapies cytotoxiques. Ceci est lié, en partie, au déclenchement d'une mort cellulaire immunogène (MCI) par certains cytotoxiques, stimulant l'adjuvanticité des cellules tumorales. La MCI de la cellule tumorale est caractérisée par l'émission de signaux de dangers permettant de favoriser le recrutement et l'activation des cellules dendritiques, ainsi que la réponse immune antitumorale adaptative médiée par les lymphocytes T. La première étape de la MCI est l'exposition de la calréticuline à la face externe de la membrane plasmique, favorisant la phagocytose par les cellules dendritiques. Ensuite, les cellules tumorales sécrètent de l'ATP dans le milieu extra-cellulaire, par un mécanisme d'exocytose lysosomale dépendant de l'autophagie, favorisant le recrutement des cellules dendritiques. Enfin à un stade tardif, les cellules tumorales mourantes libèrent d'importantes quantité de protéines nucléaires dont l'HMGB1, ce qui entrainera la maturation des cellules dendritiques. Parmi les cancers du sein, le sous-type triple-négatif (TN) est le plus agressif, mais également le plus immunogène. Les patientes présentant un cancer du sein TN métastatique peuvent désormais bénéficier d'une combinaison de chimiothérapie et d'immunothérapie (avec un inhibiteur de checkpoint anti-PD-L1), même si les résultats présentés en septembre 2020 au congrès Européen de cancérologie (ESMO) remettent en cause l'efficacité de cette association. Malgré cette combinaison, la majorité des patientes rechuteront la première année. Le développement de thérapeutiques potentialisant la réponse immune est donc un enjeu majeur. La Dendrogénine A (DDA) est un métabolite suppresseur de tumeur caractérisé par l'équipe de Marc Poirot, avec une activité cytotoxique démontrée dans le cancer du sein hormono-dépendant, le mélanome et la leucémie aigüe myéloïde. La DDA induit une mort cellulaire par le déclenchement d'une autophagie dépendante de la voie du récepteur Liver-X-receptor (LXR) ß. Durant ma thèse, nous avons montré que la DDA exerçait une activité cytotoxique dans plusieurs lignées murines in vitro et in vivo, et une lignée humaine de cancer du sein TN in vitro. Nous avons montré que la DDA induisait des marqueurs d'autophagie dans ce modèle, in vitro et in vivo. Ensuite, nous avons montré qu'un traitement par DDA déclenchait les signaux de MCI sur deux lignées de cancer du sein TN (murine 4T1, et humaine MDA-MB-231), et une lignée murine de mélanome (B16F10). Les signaux de MCI induite par la DDA étaient supérieurs à ceux obtenus avec deux cytotoxiques standard, la doxorubicine et le mafosfamide. Nous avons enfin montré in vivo qu'une vaccination de souris immunocompétentes par des cellules mourantes traitées avec de la DDA, à partir de deux lignées cellulaires distinctes (4T1 et B16F10), induisait une protection prophylactique partielle lors du rechallenge de ces souris avec des cellules tumorales viables, en dehors de tout traitement systémique. Ces résultats nous montrent que la DDA pourrait être une nouvelle thérapeutique potentialisant la réponse immune antitumorale dans le cancer du sein TN
Last decade, several pre-clinical and clinical studies well demonstrated that the efficacy of conventional chemotherapies involves an immunological component. A part of the explanation comes from the demonstration that conventional chemotherapies can boost the adjuvanticity of cancer cells by inducing an immunogenic cell death (ICD). ICD of tumour cells drive an inflammatory response characterized by the activation of dendritic cells and the initiation of a cytotoxic T-lymphocyte immunity. During ICD, the reticulum endoplasmic stress promotes the translocation of the calreticulin protein to the cell surface, that facilitates the phagocytic uptake of tumour cells by immature dendritic cells. Then, the activation of autophagy in tumor cells induces the lysosomal secretion of ATP, that promotes the recruitment of dendritic cells. Lastly, dying cancer cells release a large amount of nuclear proteins including HMGB1, that drives the maturation of dendritic cells upon binding to TLR4. TNBC is defined as the most aggressive subtype of breast cancer, classified by its lack of expression of the hormonal receptor and the human epidermal growth factor receptor 2, but also considered as the most immunogenic subtype of breast cancer. A subset of TNBC patients are now eligible for immunotherapy in combination with chemotherapy, but all of them will finally relapse, mostly during the first year of treatment. Development of novel therapeutics to optimize immune response in these patients is urgently needed. Dendrogenin A has been characterized by the Marc Poirot's team as a tumour suppressor metabolite present in normal breast tissue, but absent in neoplastic breast tumour. DDA has an anti-tumour activity demonstrated in hormone-dependent breast cancer and melanoma cells, through the induction of an LXRß-dependent autophagy. During my thesis, we showed that DDA elicit cell death and autophagy in triple-negative breast cancer (TNBC) models in vitro and in vivo. Then, we demonstrated that DDA induced hallmarks of ICD in vitro in TNBC and melanoma cells lines. Indeed, we demonstrated that a treatment with DDA trigger (1) surface exposure of CALR, (2) release of ATP in the supernatant in an autophagy-dependent manner, and (3) release of HMGB1 in the supernatant. These danger signals were induced by DDA in a larger extent than doxorubicin and mafosfamide, described as two ICD-inducers. We then demonstrated in two different models that cancer cells undergoing ICD after being treated with DDA provide partial immune-mediated prophylactic protection against a subsequent challenge with living cancer cells of the same type. These results suggested that DDA could be a new therapeutic developed to potentiate antitumoral immune response in TNBC

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32

Mothe, Pujadas Beatriz. "Rational Design and Testing of Novel HIV T Cell Immunogens". Doctoral thesis, Universitat Autònoma de Barcelona, 2012. http://hdl.handle.net/10803/96703.

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Aquesta tesi doctoral té com a objectiu caracteritzar millor les respostes VIH específiques de les cèl·lules T (CTL), identificar les dianes virals implicades en el control del virus i desenvolupar com aquesta informació s'ha incorporat en el disseny d’un inmunogen per a una futura vacuna contra el VIH. S’inicia amb una introducció actualitzada i àmpliament referenciada sobre el VIH/sida, inclosos aspectes històrics i epidemiològics de l’epidèmia, així com una visió des de la virologia i la immunologia de les dificultats en el desenvolupament de vacunes eficaces contra el VIH. En el primer capítol, es revisa la interacció entre factors virals, característiques genètiques de l’hoste i mecanismes immunològics implicats en el control del VIH. S’han incorporat les troballes més recents sobre aspectes immunològics -especialment en CTL- correlacionats amb l’evolució de la malaltia. A més, s’exposen els nous coneixements sobre com l'evolució viral és modula per diferències en la freqüència de característiques genètiques de l'hoste en les diferents regions del món. Es desenvolupa alhora la importància de discernir millor entre causa i efecte dels marcadors immunològics associats amb el control relatiu de la infecció pel VIH, doncs els factors que són un mer reflex de la replicació continguda del virus per altres motius poden induir a errors de concepte que s’incorporin al disseny de noves vacunes. Al llarg del segon capítol, s’exposa la identificació de 26 dianes virals (OLP) del VIH més potents per induir respostes CTL associades amb un millor control de la replicació viral en tres cohorts molt àmplies de pacients infectats. Es demostra que l'efecte dels OLP identificats en les càrregues virals dels pacients és tan fort com l'associat a la influència de l’ HLA individual. Alhora, les dianes identificades com a beneficioses van resultar trobar-se en regions més conservades dins del genoma viral, fet que està en relació amb treballs d’altres grups enfocats en els elements conservats del virus. Una contribució important del nostre treball és la incorporació d'una quantitat significativa de dades d’ immunogenicitat real, ja que no totes les regions conservades del proteoma viral són igualment immunogèniques i per tant, no totes poden considerar-se igualment beneficioses. En el tercer capítol, es va associar l’avidesa funcional i la capacitat de reaccionar amb variants virals amb el control del VIH. En aquest treball, es va incloure un grup d'individus infectats pel VIH amb un fenotip de 'controlador' i ‘progressor’ del que es van excloure deliberadament tots els participants que expressaven alguns dels HLA prèviament coneguts com a favorables en la infecció per VIH, per tal d’evitar el possible biaix a causa de la dominància de CTLs dirigides a epítops restringits per aquests al·lels beneficiosos. La idea és trobar resultats que siguin el màxim de traduïbles a la població general, on aquests HLA es presenten a unes freqüències molt baixes. El nostre treball alhora complementa una visió més amplia de la funcionalitat de les CTL. Tot i la limitació de l'avaluació d'una sola funció efectora (excreció d’IFNγ) el fet que l'avidesa es mostri estar vinculada a la reactivitat creuada, pot ser determinant per la inducció de respostes amb gran ‘profunditat’ (reactivitat creuada a variants virals) i així ajudar a contenir l’escapament a la pressió immunològica així com respondre a l’enorme diversitat viral, alguns dels principals obstacles en el desenvolupament de vacunes. Finalment, tots els resultats van ser incorporats per dissenyar una nova seqüència per a un immunogen reduccionista, que té com a objectiu cobrir un ampli repertori d’HLA, trencar amb la inmunodominància de respostes induïdes a regions que no apareixen com especialment beneficioses en les cohorts que hem testat alhora que tractar d'enfocar la inducció de respostes cap a les dianes virals identificades com a més beneficioses. Es va construir un plàsmid de DNA que expressa l’inmunogen dissenyat d’una forma estable in vitro. Es presenten els primers assaigs in vivo en ratolins C57BL/6 en els que l’immunogen va induir una resposta immunològica CTL particularment amplia. Al llarg de la discussió s’elaboren el punts forts i febles dels nostres resultats en un context més ampli del camp i es descriu la orientació futura del nostre treball.
This doctoral thesis aims to better characterize HIV specific T cell responses, define the viral targets most implicated in mediating viral control and discuss how this information has been incorporated into a rational immunogen design for a future HIV vaccine. A well-researched and referenced introduction to HIV, including historical and epidemiological aspects of the infection as well as virological and immunological overview of the vaccine development field challenges is firstly provided. In the first Chapter, a review of the important interplay between viral factors, host genetic and immune characteristics in HIV control is given. Some novel insights on immune correlates –specially on CTLs- elucidated over the last years to complete the work compiled in Chapter 1 are discussed. Also, new data is presenting regarding how viral evolution is shaped by differences in the frequency of different host genetic markers within different regions. New concepts to better discern between cause and effect of immune parameters associated with relatively controlled HIV infection will be discussed, as the factors that are a mere reflection of otherwise contained replication can mislead vaccine design. Throughout the second Chapter, the most potent viral targets of the virus-specific T cell responses in controlling viral replication were identified in 26 regions over the HIV proteome. The relative effects of host genetics (i.e. HLA) and CTL specificity on HIV-1 control were assessed in large cohorts of HIV infected individuals. Importantly, the effect of T cell specificity towards the identified regions on viral loads was shown to be at least as strong as the associated with host HLA genetics. These preferred targets turned out to be in the most conserved regions of the viral genome, which is in accordance with other investigators work developing novel vaccine concepts focused on conserved elements. An important contribution of our work is the incorporation of a significant amount of immunogenicity data, as not all conserved regions of the viral proteome are equally immunogenic. In the third chapter, functional avidity and the ability to react with viral variants were associated with controlled HIV infection. In the present work, a group of HIV infected individuals was chosen with a ‘controller’ and a ‘progressor’ phenotype, but those expressing some of the known favourable HLA were intentionally excluded from the cohort to avoid bias due to the presence of dominant CTL epitopes restricted by these beneficial alleles and find results more translatable to the general population. Our work complements recent data trying to have a bigger picture of CTL functionality. Despite the limitation of assessing a single effector function (IFNγ release) the fact that functional characteristics such as avidity are shown to be linked to cross-reactivity, can be determinant for the depth of vaccine induced T cell responses and therefore, help in dealing with escape and viral diversity, some of the major hurdles in vaccine development. Lastly, all the compiled findings were incorporated into a new reductionistic HIV immunogen sequence with a broad HLA heterogeneity restriction that aims to break immunodominance to regions with potential non-beneficial effects in viral control and seek to focus the vaccine induced response to most protective viral targets. A DNA plasmid expressing the HIV T cell immunogen was first engineered , showed to have a stable expression in-vitro and induced a particularly broad T cell responses in the first in vivo immunogenicity studies in C57BL/6 mice. Strong and weaker points of our findings are elaborated in a larger context of the field and next future directions of our work are outlined throughout the Discussion of this Thesis.

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Liljeqvist, Sissela. "Recombinant subunit vaccines : protein immunogens, live bacteria and nucleic acids /". Stockholm : Tekniska högsk, 1998. http://www.lib.kth.se/abs98/lilj0520.pdf.

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Donovan, Elizabeth Anne. "Identification and characterisation of immunogenic vaccine candidates of 'Actinobacillus pleuropneumoniae'". Thesis, University of Nottingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435764.

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Pineo, Catherine. "Plant production and immunogenic characterisation of Human papillomavirus chimaeric vaccines". Master's thesis, University of Cape Town, 2011. http://hdl.handle.net/11427/12240.

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Includes abstract.
Includes bibliographical references (leaves 153-175).
Cervical cancer is primarily caused by infection with Human papillomavirus (HPV) and is a global concern, particularly in developing countries which contain ~80% of the cervical cancer burden. Current HPV L1 major capsid protein virus-like particle (VLP)-based vaccines are effective in the type-specific prevention of infection and associated disease. However, the high cost of the vaccines has limited their widespread application, and cytological screening programmes are still required to detect malignant lesions associated with the non-vaccine types, particularly in HIV-infected populations.

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Thompson, Riley Jacob. "Discovery and Evaluation of Immunogenic Antigens for Bovine Brucellosis Serodiagnostics". Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42349.

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Brucella spp. are zoonotic infectious agents, primarily of livestock, that cause the disease brucellosis. Bovine brucellosis, caused by Brucella abortus, is of greatest concern due to the disease’s significant economical and public health impact. Canada fully eradicated bovine brucellosis from domesticated cattle herds in 1985, however, continued surveillance through screening for B. abortus exposure is paramount to the maintenance of bovine brucellosis eradication nationwide. The Canadian Food Inspection Agency (CFIA) is responsible for the surveillance of bovine brucellosis outbreaks in Canada and the maintenance of eradication. Current B. abortus serodiagnostics and serological screening is mostly based on the detection of antibodies against Brucella lipopolysaccharide (LPS), a highly immunogenic component of the outer cellular membrane. Such tests face difficulties with false positive results due to cross reactivity with other Gram-negative bacteria that produce LPS. The purpose of the research presented here was to address this issue through identifying new B. abortus protein antigens for the improvement of serological test specificity. In this study, 101 candidates were identified through predictive bioinformatic analyses and selected for immunogenic evaluation. While none of the expressed candidates displayed positive serological activity with in-house brucellosis positive bovine serum panels, the workflow presented here can be used for continued research and the assessment of more proteins from B. abortus and other bacterial pathogens.

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Behrendt, Rayk. "Immunogene und immunsuppressive Eigenschaften des transmembranen Hüllproteins gp41 von HIV". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15993.

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Die Entwicklung eines effektiven HIV-Impfstoffes ist bis heute nicht gelungen.Konventionelle Immunisierungsstrategien mit rekombinant hergestellten Hüllproteinen des Virus in verschiedensten Formen induzierten keine subtypenübergreifende, protektive Immunantwort gegen HIV. Die Gewinnung und Charakterisierung der gp41-spezifischen breit neutralisierenden monoklonalen Antikörper 2F5 und 4E10 bildete die Grundlage einer Reihe neuer epitopgerichteter Ansätze für die HIV-Impfstoffentwicklung. Bisherige Immunisierungsstudien basierten auf der Verwendung des linearen Hauptepitopes (E2) der beiden Antikörper aus dem C-terminalen Teil der Ektodomäne von gp41. Nach neueren Erkenntnissen, reicht für eine effektive Neutralisation durch 2F5 oder 4E10 die Bindung dieser Antikörper an ihr lineares Epitop in der membran proximalen externen Region (MPER) von gp41 allein nicht aus. Vielmehr wurde die Beteiligung einer N-terminalen Domäne (E1) von gp41 an der neutralisationsaktiven Bindung von 2F5 bzw. 4E10 postuliert. In dieser Arbeit wurden die beiden 2F5 und 4E10 spezifischen Epitopbereiche E1 und E2 des gp41 erstmals in das strukturell verwandte transmembrane Hüllprotein des Koala Retrovirus (KoRV) eingebracht. Die Applikation der hergestellten Antigene erfolgte sowohl in Form der codierenden DNA mittels ballistischer Immunisierung (GeneGun®) als auch durch bakteriell exprimierte Proteine. Mit beiden Strategien konnten für drei Hybridproteine in den ersten Studien eine HIV-1 gp41 spezifische, breit neutralisierende humorale Immunantwort induziert werden. Diese Ergebnisse konnten jedoch in späteren Studien nicht reproduziert werden. Die Analyse der induzierten Immunantworten zeigte eine Verlagerung der Hauptimmunantwort als deren Ursache eine bakterielle Fremdinfektion der Versuchtiere diskutiert wurde. Zur Evaluierung der Immunisierungsstudien wurde ein neuartiger real time PCR basierter in vitro Neutralisationstest um Kontrollen zur Virusspezifität und Cytotoxizität erweitert.
The development of an effective HIV vaccine is considered the to play a key role in controlling the HIV pandemic. Conventional immunisation strategies using recombinant envelope proteins of the virus did not lead to the induction of a broad range protective immunity. A new target sequence for the induction of a broadly neutralising humoral immune response has been discovered through the characterization of the gp41 specific broadly neutralising monoclonal antibodies 2F5 and 4E10. Until now all attempts to induce 2F5/4E10 like neutralising antibodies failed. So far only the linear main epitope (E2) of 2F5 and 4E10, located in the C-terminal part of the gp41 ectodomain was used as the target sequence. However, it was recently shown that an N-terminal domain (E1) of gp41 increases the avidity of 2F5 to its epitope. The E1 domain may therefore be involved in the mediation of a neutralisation active binding. For the first time immunisation strategies have been developed that target both previously identified domains (E1 and E2) of gp41. The sequences corresponding to E1 and E2 have been introduced at homologous positions in the structurally related transmembrane envelope protein p15E of the Koala Retrovirus (KoRV). These generated hybrid antigens have been used for immunisation of wistar rats. They were applied as recombinant proteins expressed in E.coli and as DNA using a ballistic immunisation (GeneGun®) approach. Although in first trials neutralising antibodies specific for gp41 of HIV-1 were induced, these results could not be reproduced. Analysis of the induced antibodies showed a shift of their binding specifity. A bacterial infection of the used animals was identified as the cause of the unexpected shift in the antigen specific humoral immune response. For evaluation of the immunisation studies a new neutralisation assay based on the measurement of provirus integration by duplex real time PCR has been extended for controls of virus specifity and cytotoxicity.

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Beena, T. K. "Antigenic Determinants Of Chicken Riboflavin Carrier Protein: Structural And Functional Aspects". Thesis, Indian Institute of Science, 1994. http://hdl.handle.net/2005/141.

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Investigations detailed in this thesis constitute a part of the continuing programme of research undertaken in our laboratory on the riboflavin carrier protein (RCP) with particular reference to identification and synthesis of neutralizing antigenic determinants, design of relevant epitope mimetics with improved immunogenic characteristics and relationship between their secondary structures and immunological properties. The riboflavin carrier protein is elaborated as a reproductive stratagem to ensure adequate vitamin deposition in the developing oocyte in the chickens. The protein is scrupulously conserved through evolution in terms of physico chemical and immunological characteristics from fish through birds to mammals, including primates. In rodents and subhuman primates immunization with the heteroantigen viz., chicken egg white RCP leads to functional neutralization of the endogenous maternal protein resulting in curtailment of early pregnancy. Thus, the crucial role of RCP in maintenance of pregnancy is established and the protein identified as a potential candidate vaccine for immimocontraception. Further studies with the reduced and carboxymethylated (RCM) RCP as the immunogen reveal that antibodies induced by RCM-RCP are equally effective in bioneutralization of the endogenous protein. So it can be surmised that the native folded structure of RCP is not obligatory for eliciting bioneutralizing antibodies. In an attempt to identify functionally relevant regions of the protein, a panel of monoclonal antibodies (MAbs) have been raised and characterized. One of the MAbs viz., 6J32Ci2 could bring about early fetal resorp-tion when injected to mice with confirmed pregnancies. These results prompted a detail molecular immunological approach to understand underlying mechanisms. The principal aims of the present investigations include: (1) identification of neutralizing epitopes; (2) synthesis of peptidyl sequences incorporating these determinants; (3) an understanding of the structure, antigenic and immunogenic characteristics of these peptides; (4) correlation of conformational and antigenic characteristics; (5) rational design and synthesis of peptide analogs with greater propensity to assume predicted secondary structures; (6) analysis of conformation dependency of peptide antigens and the importance of such conformation in generating an optimal B-cell response; (7) the efficacy of the antibodies elicited by these Peptide antigens in neutralizing endogenous protein with the ultimate aim of designing synthetic vaccines. Chapter 1 of this thesis deals with a general introduction summarizing the current status of knowledge regarding the chemistry and biology of RCP as well as synthetic peptides as potential immunogens. Chapter 2 outlines details of the experimental procedures adopted. Chapter 3 describes the results of investigations on the C-terminal fragment (residues 200-219) of cRCR The main consideration in selecting this sequence for the design of a potential peptide-based vaccine relied on the epitopic specificity of the neutralizing MAb 6S2C12. Epitope mapping using the Pepscan method revealed that the monoclonal antibody recognizes a core sequence corresponding to residues 203-210 of the cRCP. A 21-residue synthetic peptide (C-21) comprising this epitope was synthesized and antibodies elicited to the peptide conjugated to two different carriers, namely diphtheria toxoid and purified protein derivative (PPD) for T-cell help. In both active and passive immunoneutralization experiments, the peptide specific neutralizing antibodies interfered with the biological function of the protein and hence either protected from pregnancy or caused early fetal resorption in rodents as well as in sub-human primates. The conforma-tional properties of the peptide in aqueous buffers were analyzed from circular dichroism which revealed the absence of any ordered structure in the native C-21 peptide. Theoretical predictions of secondary structure suggested a propensity for an t*-helical structure for this fragment in the native protein. Therefore, influence of the helix-promoting solvent, vizM 2,2,2,trifluroethanol (TFE) on the C-21 peptide was investigated. Addition of TFE resulted in spectral changes with negative bands at 208 and 222 nm and a positive band at 190 rim which are typical of an a-helix. To gain more information on the conformational characteristics of this peptide, it was considered worthwhile to stabilize the native peptide in an a-helical conformation based on simple rational design principles. Towards this end, four analogs of the parent peptide were synthesized and helix stabilization was sought to be achieved by introducing either salt bridges or back-bone conformational constraints such as by incorporating a-amino isobutyric acid at appropriate positions. In all the analogs, the core sequence, recognised by the neutralizing MAb 6B2C12 was maintained intact to ensure induction of antibodies capable of recognizing the native protein. CD spectral analysis of the analog peptides indicated that all the engineered peptides had varying degrees of enhanced helicities as compared to the parent peptide. The immunogenicity of each analog was studied by to the relevant peptide-diphtheria toxoid conjugates and analyzing their reactivities with the native protein by direct and competitive ELISA. The results revealed that these engineered conformational analogs axe highly immunogenic eliciting high titers of anti-protein antibodies. The relative affinities of these antibodies to bind cRCP were investigated. The antibodies to peptide analogs had higher affinities for the native protein and a positive correlation was found between the helical content of the peptide antigen in question and the relative affinity of corresponding antibody. The antibodies directed to all the peptide analogs could block the function of RCP resulting in early embryonic resorption when administered to pregnant mice. An interesting pattern of immunological cross-reactivity has been observed with the native and designed peptides. Antibodies raised to constrained helical analogs could bind the C-21 peptide which is structurally flexible. In contrast, the antibodies raised to the flexible native peptide antigen were inefficient in recognizing the structured peptides. The ability of all the peptide antibody to bind the native protein has been interpreted in terms of a conformationally flexible C-terminus region in cRCP. Chapter 4 details investigations on a 21-residue peptide (N- 21) from the N-terminiis (4-24) of the protein. Selection of this peptidyl sequence relied on theoretical prediction of potential sequential determinants on RCP other than at C-terminus as well as on the outcome of immunoneutralisation experiments using antibodies to egg yolk RCP which lacks the relevant C-terminal determinants. The structure of this peptide in solution was analyzed by two dimensional NMR and CD. NMR experiments revealed the presence of two structured regions in the peptide. Diagnostic nuclear Overhauser effects characteristics of reverse turns or short frayed helical segments over residues 3-9 and 18-21 of the peptide were obtained. CD spectra showed the presence of a strong, negative band at 204 nm over a wide range of solvent conditions, a feature which has been interpreted in terms of a "polyproline Il-like" segment encompassing residues 11-16 which corresponds to an interesting (X-Pro)^ repeat in the N-21 sequence. Specific antibodies were generated to this peptide as a conjugate with diphtheria toxoid. Administration of the antipeptide antibodies could neutralize the protein in vivo as demonstrated by early embryonic loss in pregnant mice. In limited experiments the antipeptide antibodies showed propensity to protect bonnet monkeys from pregnancy over a few consecutive ovulatory cycles when titres are maintained elevated by periodic boosting. To address the relationship between peptide structures and antigenicity, epitope mapping of this antipeptide antibodies as well- as the polyclonal antibodies to native RCP was undertaken using the Pepscan method. The results reveal that antigenic regions correspond well to conformationally well-defined elements of structure with the polyproline II-like segment being a common antigenic determinant on both the peptide and the native protein. These observations are suggestive of the involvement of both the N and C-terminal regions of RCP in terms of its binding to putative plasma membrane receptors.

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Altindis, Emrah. "Identification Of The New Immunogenic Proteins Of Bordetella Pertussis By Immunoproteomics". Master's thesis, METU, 2007. http://etd.lib.metu.edu.tr/upload/12608320/index.pdf.

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The genus Bordetella contains several pathogenic species generally associated with upper respiratory tract infections in warm-blooded animals. Bordetella pertussis is the etiologic agent of whooping cough. Whooping cough is presently one of the ten most common causes of death from infectious diseases and reported by the World Health Organisation (WHO) to cause 50 million cases and 350000 deaths worldwide per year, mainly among unvaccinated individuals in poor countries. The term proteome, in analogy to the term genome, was coined to describe the complete set of proteins that an organism has produced under a defined set of conditions. Proteomics has been used to identify novel bacterial vaccine candidates against several human pathogens. Fueled by growing DNA sequence information, the analysis of the proteome becomes a valuable and useful tool for antigen discovery. Much of information about immunogenic component can be derived from proteomics coupled to Western blotting, namely immunoproteomics. v In the present study, we report first immunoproteomics analysis to identify candidate antigens of B. pertussis for vaccine development. Different sera from mice, which were immunized or challenged with B. pertussis, were analyzed for reactivity by Western blot against whole cell extracts of B. pertussis Tohama and Saadet strains separated by 2-DE. We identified 15 immunogenic proteins of Bordetella pertussis as a total (60 kDa chaperonin, heat shock protein, serum resistance protein, putative substrate-CoA ligase, ATP-dependent protease, preprotein translocase secA subunit, S-adenosylmethionine synthetase, elongation factor Tu, RNA polymerase alpha subunit, ketol-acid reductoisomerase, pertactin, lysyl-tRNA synthetase, serum resistance protein, carbamoyl-phosphate synthase large chain, 30S ribosomal protein S1 subunit), 6 of which being identified as immunogenic in a pathogenic microbe (ATP-dependent protease, carbamoylphosphate synthase large chain, lysyl-tRNA synthetase, putative chromosome partition protein, preprotein translocase secA subunit, 30S ribosomal protein S1 subunit) and 5 identified as immunogenic for Bordetella pertussis (RNA polymerase alpha subunit, S-adenosylmethionine synthatase, putative substrate-CoA ligase, elongation factor Tu, ketol-acid reductoisomerase) for the first time.

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Cortez-Gonzalez, Xochitl. "Immunogenic peptides of human telomerase reverse transcriptase restricted to HLA-B7 /". Diss., Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3266847.

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Roy, Amrit [Verfasser]. "Immunogene Wirkung des TEL/AML1 Onkopeptids auf leukämische Zelllinien / Amrit Roy". Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2016. http://d-nb.info/1121007635/34.

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Watson, Douglas Stuart. "Lipopeptide immunogens targeting the membrane proximal region of HIV-1 gp41". Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3359565.

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Thesis (Ph. D.)--University of California, San Francisco with the University of California, Berkeley, 2009.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3649. Adviser: Francis C. Szoka.

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43

Wantz, May. "Evaluation of innovative platinum compounds as antitumor agents combining chemotherapy and immunotherapy". Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ053.

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Les agents chimiothérapeutiques à base de platine (II) actuellement utilisés en clinique ont de nombreux effets secondaires limitant ainsi leur utilisation. Voilà pourquoi, nous avons décidé de développer des composés innovants à base de platine : les carbènes N-hétérocycliques liés au platine et associés au polyéthylèneimine. Nous avons évalué leur activité cytotoxique ainsi que leur effet sur le système immunitaire étant donné que certains dérivés de platine commercialisés sont capables d’activer le système immunitaire. Après avoir testé différents conjugués, nous avons sélectionné un candidat qui présente un profil cytotoxique important in vitro et in vivo, tout en limitant les effets secondaires : le NHC-Pt(II)-PEI30. Ensuite, nous avons pu montrer que ce complexe était également capable de favoriser l’apoptose de cellules souches cancéreuses de glioblastome, des cellules responsables de la réapparition de plusieurs cancers et résistantes à de nombreux traitements anticancéreux. Finalement, nous avons constaté que le NHC-Pt(II)-PEI30 seul induisait seulement une faible mort immunogène des cellules cancéreuses, limitant ainsi l’activation du système immunitaire. Voilà pourquoi, nous avons associé notre composé de platine à un adjuvant pour augmenter la réponse immunitaire antitumorale. Ces résultats suggèrent que notre composé innovant à base de platine (II) constitue un outil intéressant pour combiner la chimiothérapie et l’immunothérapie
Platinum(II)-based chemotherapeutic agents, which are actually used in clinics, have numerous side effects, thus limiting their use. That is why, we decided to develop innovative platinum compounds: N-heterocyclic carbene-platinum complexes associated with polyethylenimine. We assessed their cytotoxic activity and their effect on the immune system, as some commercially available platinum derivatives are able to activate the immune system. After evaluation of various conjugates, we selected one candidate displaying an important cytotoxic profile in vitro and in vivo, but with few side effects: NHC-Pt(II)-PEI30. Moreover, we showed that this complex was able to induce apoptosis of glioblastoma-derived cancer stem cells, which are resistant to numerous anticancer therapies and are responsible for cancer relapse. Finally, we observed that NHC-Pt(II)-PEI30 alone only induced a weak immunogenic cell death, limiting this way the activation of the immune system. That is why, we associated our platinum compound with an adjuvant in order to enhance the antitumor immune response. These results suggest that our innovative platinum(II) compound displays interesting properties for the combination of chemotherapy and immunotherapy

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Kerrigan, Sarah. "Immunochemical detection of lysergic acid diethylamide using a photochemically linked immunogen". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/nq27177.pdf.

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Wiedemann, Maximilian. "Untersuchungen zum Spektrum immunogener Proteine bei Encephalitozoon cuniculi". Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-60918.

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Derby, Nina Rafterman. "Designing immunogens to elicit broadly reactive neutralizing antibodies to the HIV envelope /". Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/9302.

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Rojas, JoseÌ-Manuel. "Identification of novel immunogenic HLA-DR-restricted peptides from tumour-associated antigens". Thesis, Nottingham Trent University, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273771.

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CD4+ T cells playa central role in antitumour immunity; not only do they provide help for the development of CTL recognising tumour antigens but they can also enhance antitumour responses via indirect cytotoxic mechanisms at the tumour site. Since CD4+ T cells recognise the antigen in the form of pep tides presented on MHC class II molecules, attention has been focused in the recent years on the identification of these peptides derived from tumour antigens. Therefore the aim of this study was to identify novel immunogenic peptides derived from tumour antigens where presentation was restricted to human MHC class II HLA-DRI and/or HLADR4 molecules. The adopted strategy consisted in predicting peptides from the tumour antigens p53, gplOO and bcr-abl(b3a2) using computer-assisted algorithms, and immunisation of HLA-DRI transgenic mice with these peptides in order to assess their immunogenicity. Immunogenic peptides in transgenic mice were then tested in human in in vitro T cell sensitisation assays. To determine peptide immunogenicity in mice, a method was optimised using the reported I-Ak-restricted peptides HEL46-61 and HEL1l 9-132. This model was then successfully established in HLA-DRI transgenic mice with the model peptide HA307- 319. Proliferative responses and IFN-y production were observed when the splenocytes of HLA-DRI transgenic mice were re-presented in vitro with the HA307-319 peptide used in immunisation. Dendritic cells (DC) were shown to be better antigen presenting cells (APC) than syngeneic splenocytes in proliferation assays; thus DC were subsequently used as APC in the all experiments. Further characterisation of DC, generated from bone marrow precursors by culture with GM-CSF, demonstrated that day 8 non-adherent cells matured with LPS were optimal for antigen presentation in this experimental setting. Immunisation of HLA-DRI transgenic mice with predicted peptides from p53, gplOO and bcr-abI(b3a2) resulted in HLA-DRlrestricted responses for two novel p53 peptides (p5363-77 and p53108-122) and two bcrabl peptides (bcr-abIGFK11 and bcr-abIATG1 8). Responses were also observed to two novel gp 100 peptides (gp 1 00194-208 and gp 100566-580). This study demonstrated that HLA-DR-restricted responses to novel peptides can be obtained in HLA-DRI transgenic mice. Furthermore, proliferative responses to p5363-77 in a HLA-DRI + donor, to gpl00566-580 in another HLA-DRl+ donor. and to p53108-122 in two HLA- -1- Abstract DR4+ donors demonstrated that these peptides were also immunogenic in human assays. Collectively, these results indicated that peptide immunisation of HLA-DRI transgenic mice could facilitate the identification of novel immunogenic HLA-DRrestricted pep tides from tumour antigens, that allow us to understand further the role of CD4+ in antitumour immunity and improve cancer immunotherapeutic strategies.

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48

Winterling, Karina [Verfasser], Harald [Akademischer Betreuer] Kolmar y Jörg [Akademischer Betreuer] Schüttrumpf. "Immunogenic Determinants of Coagulation Factor VIII / Karina Winterling ; Harald Kolmar, Jörg Schüttrumpf". Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2018. http://d-nb.info/1162275197/34.

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Hassan, Hoda Abdel-Hadi. "Identification and characterisation of app : an immunogenic autotransporter protein of Neisseria meningitidis". Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368253.

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Lambert, Laurens J. (Laurens Johannes). "Development and characterization of immunogenic genetically engineered mouse models of pancreatic cancer". Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/129020.

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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2020
Cataloged from student-submitted PDF of thesis. Vita. Page 191 blank.
Includes bibliographical references.
Insights into mechanisms of immune escape have fueled the clinical success of immunotherapy in many cancers. However, pancreatic cancer has remained largely refractory to checkpoint immunotherapy. To uncover mechanisms of immune escape, we have characterized two preclinical models of immunogenic pancreatic ductal adenocarcinoma (PDAC). In order to dissect the endogenous antigen-specific T cell response in PDAC, lentivirus encoding the Cre recombinase and a tumor specific antigen (SIINFEKL, OVA[subscript 257-264]) was delivered to Kras[superscript LSL-G12D/+]; Trp[superscript 53flox/flox] (KP) mice. We demonstrate that KP tumors show distinct antigenic outcomes: a subset of PDAC tumors undergoes clearance or editing by a robust antigen-specific CD8+ T cell response, while a fraction undergo immune escape. Subsequently, we have developed an immunogenic pancreatic tumor organoid orthotopic transplant model.
In this model, immunogenic pancreatic tumors manifest divergent tumor phenotypes; 40% of tumor organoids do not form tumors ("non-progressors"), whereas 50% of organoids form aggressive tumors despite maintaining antigen expression and a demonstrable T cell response ("progressors"). Additionally, a subset (10%) of tumors show an intermediate phenotype, possibly reflective of an immune equilibrium state. We have further phenotypically and transcriptionally characterized the CD8+ T cell response to understand immune escape in this model. Our analyses reveal unexpected T cell heterogeneity, and acquisition of T cell dysfunctionality. Therapeutic combinatorial targeting of co-inhibitory receptors identified on dysfunctional antigen-specific CD8+ T cells led to dramatic regression of aggressive pancreatic tumors.
Finally, we demonstrate that human CD8+ T cells isolated from pancreatic tumors co-express co-inhibitory receptors, suggesting that T cell dysfunction may be operational in human disease. This is the first demonstration of immunoediting in an autochthonous and organoid-based model of pancreatic cancer. Further characterization of these preclinical model systems will enable rational design of novel clinical immunotherapeutic strategies for treatment of this devastating disease.
by Laurens J. Lambert.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology

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