Malaria is one of the major infectious diseases in the world with regard to mortality and morbidity, and the development of a vaccine against the malaria parasite Plasmodium falciparum is considered of high priority. The aim of the work presented in this thesis was to develop and characterize recombinant vaccine constructs based on the P. falciparum asexual blood-stage antigen Pf332. We have studied the humoral responses in mice elicited by various types of constructs, including naked DNA plasmids, naked mRNA, alphavirus, and peptides. Immunological memory was successfully induced against the repetitive EB200 fragment of Pf332, although the antibody titers were generally low and the highest titers were unexpectedly obtained with a conventional DNA plasmid. In another study, we also demonstrated the ability to circumvent genetically restricted immune responses in mice against two malaria epitopes, one of them derived from Pf332, by inclusion of universal T-cell epitopes into multiple antigen peptide constructs. However, the overall variability of the responses stressed the importance of including several epitopes in a future malaria vaccine. Further, the recent completion of sequencing of Pf332 enabled us to identify and characterize the immunogenic properties of a non-repeat fragment of the Pf332, termed C231. Our analyses of C231 showed that antibodies raised against the recombinant protein possess an in vitro parasite inhibitory capacity similar to that of antibodies against recombinant EB200. Furthermore, the recognition of C231 by antibodies in sera from individuals naturally primed to P. falciparum, correlated well with that previously observed for the corresponding sera and EB200. When analyzing the IgG subclass distribution of anti-C231 antibodies, we noted a bias towards IgG2 and IgG3 relative to IgG1, differing from the subclass profiles of IgG binding crude P. falciparum antigen, which were dominated by IgG1. Taken together, the work presented herein is likely to facilitate further studies on Pf332 as a potential target for protective immune responses, and amounts to a small step towards the realization of a malaria vaccine.

Τα MHC τάξης I πεπτίδια περιορισμένα μόνο σε καρκινικό ιστό, τα οποία παράγονται από πρωτεΐνες του ίδιου του οργανισμού υπερεκφραζόμενες από μία μεγάλη ποικιλία ανθρώπινων κυττάρων νεοπλασιών, είναι δυνητικοί στόχοι για CTL (κυτταροτοξικά Τ λεμφοκύτταρα - Cytotoxic T lymphocyte), στα οποία βασίζεται η ανοσοθεραπεία πολλών ειδών καρκίνου. Ωστόσο, δεδομένου ότι παρουσιάζονται και από τα φυσιολογικά κύτταρα μπορεί να αναπτυχθεί επίσης θυμική και / ή περιφερική ανεκτικότητα. Η αυτό-ανοχή αφορά Τ λεμφοκύτταρα ειδικά για πεπτίδια υψηλής συγγένειας προς το MHC. Αντίθετα, τα Τ κύτταρα που αντιδρούν με όγκους, κατευθύνεται εναντίον πεπτιδίων όγκων με χαμηλή συγγένεια προς το MHC. Το μεγαλύτερο εμπόδιο για τη χρήση τέτοιων πεπτιδίων για ανοσο¬θεραπεία, είναι ότι αυτά είναι ασθενώς ή καθόλου ανοσογόνα. Αυτό οφείλεται στην αδυναμία τους να σχηματίζουν μεγάλο αριθμό σταθερών συμπλοκών MHC/πεπτιδίων στην επιφάνεια των ACP (κύτταρα που παρουσιάζουν αντιγόνα, Antigen Presenting Cells). Στην παρούσα ερ¬γασία περιγράφουμε μία προσέγγιση η οποία επιτρέπει την επαγωγή CTL απόκρισης έναντι χαμηλής συγγένειας HLA Α2.1 περιορισμένων πεπτιδίων. Αυτή συνίσταται στην τροποποίηση της πεπτιδικής αλληλουχίας με την εισαγωγή στη θέση 1 μίας Tyr, η οποία ευνοεί την αλληλεπίδρα¬ση πεπτιδίου/HLA Α2.1. Χρησιμοποιήθηκαν 25 πεπτίδια με διάφορες συγγένειες και ικανότητες σταθεροποίησης με το HLA Α2.1. Η αξιολόγηση της ικανότητα τους να επάγουν CTL απόκριση έδειξε μία αυστηρή συσχέτιση μεταξύ της συγγένειας σύζευξης/ικανότητας σταθεροποίησης και της ανοσογονικότητας. Τα ανάλογά τους με Ρ1Tyr παρουσίασαν υψηλότερη συγγένεια 1,5 έως 55,5 φορές και σταθεροποίησαν το HLA Α2.1 για τουλάχιστον 2 ώρες περισσότερο από τα α¬ντίστοιχα φυσικά πεπτίδια. Επιπλέον, η Ρ1Tyr τροποποίηση δεν μετέβαλε την αντιγονική ειδικό¬τητα των πεπτιδίων, αφού τα ανάλογα με Ρ1Tyr πάντα αναγνωρίζονταν από ειδικά CTL για τα φυσικά πεπτίδια και αντιστρόφως, ειδικά CTL για τα Ρ1Tyr ανάλογα αναγνώρισαν τα φυσικά πεπτίδια. Τελικά, όπως ήταν αναμενόμενο, τα Ρ1Tyr ανάλογα των χαμηλής συγγένειας με το HLA Α2.1 πεπτιδίων νεοπλασιών ενεργοποίησαν αποτελεσματικά in vivo απόκριση ειδικών CTL για τα φυσικά πεπτίδια. / Induction of CTL response to HLA A2.1 restricted tumor peptides exhibiting a low MHC binding affinity and a weak immunogenicity. MHC class I restricted peptides derived from self-proteins overexpressed by a wide variety of human tumor cells are potential targets for a broad spectrum CTL based immunotherapy in cancer. However, since they are presented by normal cells they may also represent thymic and/or peripheral tolerogens. Self-tolerance principally concerns high MHC/affinity peptide specific T cells and the tumor reactive T cells repertoire that is available to be mobilized by peptide based tumor vaccination protocols is directed against low MHC/affinity tumor peptides. A major barrier to the use of low MHC/affinity tumor peptide for immunotherapy is that they are weakly or even not immunogenic. This is due to their inability to form a high number of stable MHC /peptide complexes on the surface of antigen presenting cells. In the present work we describe an approach that allows the induction of a CTL response against low affinity HLA A2.1 restricted tumor peptides. It consisted in modifying the peptide sequence by introducing a Y in the first position known to have a favorable effect in the peptide/HLA A2.1 interaction. Twenty five peptides with variable HLA A2.1 stabilization capacity are included in this study and the evaluation of their capacity to induce a CTL response in a HLA A2.1 transgenic, Db-/-, mb2m-/- mouse showed a strict correlation between MHC binding affinity/MHC stabilization capacity and immunogenicity. Their P1Y variants exhibited a 1.5 to > 55 fold higher affinity and stabilized the HLA A2.1 for at least 2 hrs more than the corresponding native peptides. These effects were more pronounced for the low affinity native peptides. Moreover, P1Y modification did not alter the antigenic specificity of peptides since P1Y variants were always recognized by the native peptide specific CTL and, inversely P1Y specific CTL recognized native peptides. Finally, as expected, P1Y variants of low HLA A2.1 affinity tumor peptides efficiently triggered in vivo a native peptide specific CTL response.

Made available in DSpace on 2016-12-08T16:24:21Z (GMT). No. of bitstreams: 1 PGCA15MA170.pdf: 1407687 bytes, checksum: 6d77197fef831a6cbdc6fcf9e39c8629 (MD5) Previous issue date: 2015-07-23 / For a long time, Pasterella multocida has been considered only as a secondary agent in pneumonias in swine. However, studies developed in Brazil identified different isolates of P. multocida able to operate as primary disease agent in swine. The objective of his project was to study protection aspects of a vaccine with pathogenic samples of P. multocida in experimental model with high sanity status pigs. In addition, it aims to describe the expression of immune response in swine induced by P. multocida infection, and determinate the prevalence of P. multocida in tonsils of vaccinated and not vaccinated pigs through immunohistochemistry. The study was divided into three experiments: Immunization of mice with vaccines prepared with different adjuvants , Test of two adjuvants used in vaccine with a P. multocida homologous strain in swine and Experimental challenge in swine with a P. multocida heterologous strain to the vaccine . Four adjuvants were tested in mice: Al(OH)3, ISA 206, Montanide GEL 01 and ISA 760. Two of them were selected for testing in swine: Al(OH)3 which showed better results in mice-, and ISA 760 for being an oily adjuvant. At the second experiment, although two vaccines have been effective in the disease protection, the one with the adjuvant Al(OH)3 was selected, because showed lower vaccine reaction. Eighteen pigs were distributed into three groups for the specific test of the vaccine for pathogenic samples of P. multocida. Group 1 (G1) vaccine with Al(OH)3; group 2 (G2) infection control; and group 3 (G3) negative control. After the experiment, the animals were euthanized, and samples were collected in ice - for microbiological cultivation and relative quantification of cytokines gene expression (IL1 β, IL2, TNFα) and TLR4 , and in formaldehyde, for routine histopathology and immunohistochemistry. Results showed that the vaccine prototype with adjuvant Al(OH)3 was efficient in controlling pulmonary pasteurellosis. The immune response was mediated by TLR4, with increased expression of TNFα. IL-1β and IL2 were also expressed, however, there was no significant difference between the groups. The immunostaining of P. multocida in tonsil was positive in all pigs challenged with the bacteria / Por muito tempo, Pasteurella multocida foi considerada apenas como agente secundário nas pneumonias em suínos, todavia, estudos realizados no Brasil identificaram diferentes isolados de P. multocida capazes de atuar como agente primário de doença em suínos. Esse projeto teve como objetivo estudar aspectos de proteção de uma vacina com amostras patogênicas da P. multocida em modelo experimental com suínos de alto status sanitário . E de forma paralela descrever expressão da resposta imune dos suínos induzida pela infecção da P. multocida e determinar a prevalência dessa bactéria em tonsilas de suínos vacinados e não vacinados através de imunohistoquímica. O trabalho foi dividido em três experimentos: Imunização de camundongos com vacinas preparadas com diferentes adjuvantes ; Teste de dois adjuvantes utilizados na vacinação de suínos frente ao desafio com uma cepa homóloga de P. multocida ; e Desafio experimental em suínos com uma cepa de P. multocida heteróloga à da vacina . Em camundongo foram testados quatro adjuvantes: Al(OH)3, ISA 206, Montanide GEL 01 e ISA 760, dos quais, foram selecionados dois para teste em suínos: Al(OH)3, que apresentou melhor resultado nos camundongos e ISA 760, por ser um adjuvante oleoso. Embora as duas vacinas tenham sido efetivas na proteção da doença, no segundo experimento, selecionou-se àquela com adjuvante Al(OH)3, que apresentou menor reação vacinal. No terceiro experimento realizado em suínos, foram utilizados 18 suínos em três grupos: grupo 1 (G1) - vacina com Al(OH)3, e grupo 2 (G2) controle de infeção e grupo 3 (G3) controle negativo. Após o experimento os animais foram eutanasiados para avaliação patológica, coleta de amostras em gelo para cultivo microbiológico e quantificação relativa da expressão gênica das citocinas (IL1 β, IL2, TNFα) e TLR4, para histopatologia de rotina e imunohistoquimica. Os resultados demostraram que o protótipo de vacina com adjuvante Al(OH)3 foi eficiente no controle de pasteurelose pulmonar. O perfil da expressão da resposta imune foi mediado por TLR4, com aumento da expressão de TNFα. Também foram expressos IL-1β e IL2, porém sem diferença significativa entre os grupos. A imunomarcação de P. multocida na tonsila foi positiva em todos os suínos desafiados com a bactéria, independente de serem ou não vacinados

A cromoblastomicose é uma micose subcutânea, com alto índice de morbidade, causada por fungos demácios, sendo o mais recorrente agente etiológico o Fonsecaea pedrosoi. Já foi descrito em todos continentes e é mais recorrente em países de clima quente, sua incidência é de grande importância no Brasil. Raramente os indivíduos se curam, pois, não há terapia padrão por conta da baixa eficácia das terapêuticas antifúngicas atuais contra a cromoblastomicose. Pouco se sabe sobre a relação parasita hospedeiro desta infeção. Os métodos de diagnóstico laboratorial se limitam a visualização das estruturas fúngicas presentes nos tecidos do hospedeiro. Decorrente da falta de informação sobre a cromoblastomicose uma doença de tamanha morbidade, surge à necessidade de estudar melhor a resposta imunológica e seus antígenos. Nosso trabalho mostrou que algumas proteínas secretadas em meio nutricionalmente pobre (exoantígenos) se mostraram imunorreativas, contra os soros de camundongos BALB/c com diferentes tempos de infecção, assim como proteínas derivadas da ruptura do fungo total através da sonicação. A proteína que se mostrou majoritariamente imunorreativa foi a de aproximadamente 50KDa, outros trabalhos descrevem proteínas de alta relevância com peso molecular próximo deste, sugerindo uma possível relação entre os achados. O sequenciamento destas bandas mostrou homologia significante com enoyl redutase, uma enzima envolvida na síntese de ácidos graxos. / The chromoblastomycosis is a subcutaneous mycosis with high morbidity, caused mostly by Fonsecaea pedrosoi, a dematious fungus. The chromoblastomycosis has been described in all continents and is more recurrent in hot climate countries, with a greatest incidence in Brazil. The host rarely heal because the therapy is low efficacy of current antifungal therapies against chromoblastomycosis. Little is known about the relationship of Fonsecaea pedrosoi with the host. The diagnostic methods is limited to visualize fungal structures, present in the host tissues. Arising from lack of information about the chromoblastomycosis, is necessary to characterize new antigens. Our work has shown that some secreted proteins in medium (exoantigens) were immunoreactive against sera from infected mice, with different times of infection, as well as proteins derived from the fungus rupture by sonication. The antigen has approximately 50 kDa. Other studies describe proteins with high relevance close this molecular weight, suggesting a possible relationship between the findings. The sequencing of these bands showed significant homology with enoyl reductase, an enzyme involved in fatty acid synthesis.

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

L’hémophilie A est une maladie hémorragique rare liée au chromosome X qui se traduit par un déficit en facteur VIII (FVIII) fonctionnel. Chez les patients atteints de la forme sévère de l’hémophilie A, l’apparition d’hémorragies incontrôlées augmente la morbidité et affecte leur qualité de vie. Le traitement de choix permettant de prévenir ou de traiter les saignements consiste en l’injection intraveineuse de FVIII thérapeutique. Cependant, chez 5 à 30% des patients, une réponse immunitaire dirigée contre le FVIII se développe. La réponse anti-FVIII est caractérisée par l’apparition d’anticorps qui inhibent l’activité pro-coagulante du FVIII. Ainsi l’apparition de ces anticorps, appelés « inhibiteurs » représente-t-elle la complication majeure du traitement des patients hémophiles A. Plusieurs facteurs de risque ont été suggérés ou proposés comme affectant le développement de la réponse anti-FVIII. Parmi eux, supporté par la théorie du « danger », la présence de médiateurs pro-inflammatoires générés par les épisodes de saignement est considérée comme adjuvant dans la réponse immunitaire anti-FVIII. Cependant, les saignements induisent de nombreux mécanismes pro mais aussi anti-inflammatoires. Ainsi, l’activation de l’endothélium vasculaire induit-elle le recrutement et l’activation des cellules effectrices appartenant aux compartiments innés et adaptatifs du système immunitaire. Au cours de ma thèse, je me suis concentré sur le rôle des saignements dans le développement de la réponse immunitaire dirigée contre le FVIII. Ainsi, ai-je découvert une association entre la présence d’un polymorphisme dans un gène induit par les saignements et la présence d’inhibiteurs du FVIII dans une cohorte de patients hémophiles A sévères. J’ai également étudié le développement de la réponse immunitaire dirigée contre le FVIII thérapeutique dans un modèle murin qui mime les saignements locaux observés chez les patients hémophiles A sévères. Finalement, j’ai caractérisé l’effet des formes réactives de l’oxygène (FRO), qui sont générées au niveau du site de saignement, sur la structure, la fonction et l’immunogénicité du FVIII. Les résultats obtenus au cours de ma thèse démontrent que les saignements ne sont pas associés à un risque plus élevé de développer des inhibiteurs dans le modèle murin largement utilisé d’hémophilie A sévère, contrairement à ce qui est communément admis. De plus, j’ai démontré une association entre un polymorphisme présent dans le promoteur du gène HMOX1 et la survenue d’inhibiteurs dans une cohorte de patients atteints d’hémophilie A sévère. En parallèle, je décris pour la première fois une altération du statut oxydatif chez les souris déficientes en FVIII et je démontre que le contrôle du statut oxydatif in vivo permet de moduler la réponse immunitaire anti-FVIII. Au contraire, l’exposition du FVIII aux FRO augmente son immunogénicité. De fait, mes résultats suggèrent que, bien que les molécules pro-inflammatoires libérées suite aux saignements puissent affecter positivement la réponse immunitaire dirigée contre le FVIII, de puissants médiateurs anti-inflammatoires sont générés in vivo et amenuisent l’effet potentiellement adjuvant des saignements. Également, ces résultats soulignent la balance complexe entre les médiateurs pro- et anti-inflammatoires qui sont générés consécutivement aux saignements ainsi que leurs effets sur la réponse immunitaire dirigée contre le FVIII thérapeutique. / Hemophilia A is a rare X-linked hemorrhagic disease consecutive to the lack of functional pro-coagulant factor VIII (FVIII). In patients with the severe form of hemophilia A, uncontrolled hemorrhages increase the morbidity and affect the quality of life. To prevent or treat bleeding episodes, therapeutic FVIII is administered intravenously. However, an anti-FVIII immune response develops in 5 to 30% of the patients. The anti-FVIII immune response is characterized by the development of anti-FVIII IgG antibodies that inhibit FVIII activity. These antibodies, referred to as “FVIII inhibitors” represent the major complication in the treatment of hemophilia A patients. Several risk factors have been suggested or proposed to predispose to the development of FVIII inhibitors. Amongst them, inspired by the “danger” theory, the presence of inflammatory mediators released upon bleeding episodes is thought to adjuvant the anti-FVIII immune response. Bleeding episodes trigger several pro and anti-inflammatory mechanisms. Thus, damage to the endothelium triggers the recruitment and activation of effector cells from the innate and adaptive compartments of the immune system. During my PhD, I investigated the role of bleedings in the development of the anti-FVIII immune response. Thus, I discovered an association between a polymorphism in the promoter region of a gene that is induced in response to bleedings, and the presence of FVIII inhibitors in a cohort of patients with severe hemophilia A. I followed the development of the immune response to therapeutic FVIII in a mouse model that mimics the localized bleedings found in patients with severe hemophilia A. Ultimately, I characterized the effects of reactive oxygen species (ROS) that are potentially released at the bleeding site in view of the structure, function and immunogenicity of the FVIII molecule. The results I obtained during my PhD demonstrate that bleedings are not associated with a higher risk for FVIII inhibitor development in the commonly used mouse model of severe FVIII deficiency. Additionally, I demonstrated an association between a polymorphism in the promoter of the HMOX1 gene and the presence of FVIII inhibitors. In parallel, I report for the first time an exacerbated oxidative status in FVIII-deficient mice as compared to control mice, and demonstrate that the control of the oxidative status in vivo reduces the anti-FVIII immune response. Conversely, the exposure of FVIII to ROS ex vivo increases the immunogenicity of the FVIII molecule. Taken together, my results suggest that, although the pro-inflammatory molecules released upon bleeding may positively affect the anti-FVIII immune response, several strong anti-inflammatory compounds are generated in vivo that dampen the potential adjuvant effect of bleedings. Ultimately, these results highlight the complex balance between the pro and anti-inflammatory mediators generated upon bleeding and their effect on the anti-FVIII immune response.

Un inconvénient majeur de l’utilisation des protéines thérapeutiques est leur immunogénicité,c'est-à-dire le déclenchement chez les patients d’une réponse immunitaire, avec production d’anticorps (anti-drug antibodies, ADA). Parmi les facteurs contributifs, les agrégats de protéines dans les spécialités administrées pourraient jouer un rôle majeur dans l’immunogénicité. Par ailleurs, la présence d’ADA de haute affinité et de divers isotypes suggère la mise en place d’une réponse immunitaire classique, faisant intervenir les cellules présentatrices d’antigènes et plus particulièrement les cellules dendritiques.Nous avons développé un modèle d’étude de l’impact d’agrégats de protéines sur la maturation de cellules dendritiques, dérivées de monocytes isolés du sang(moDC). Dans ce but, des agrégats d’hormone de croissance (GH) et d’anticorps (Rituximab) ou d’IgG1 polyclonale ont été produits et caractérisés.Nous avons montré que ces agrégats induisent la maturation des moDC, objectivée par une augmentation de l’expression de marqueurs d’activation et de co-stimulation (CD40, CD80,CD83, CD86 et HLA-DR), et par l’augmentation dela production de cytokines et chimiokines proinflammatoires (IL-6, IL-8,IL-12p40, CCL2, CCL3,CCL4 et CXCL10).En utilisant un modèle de co-culture allogénique,nous avons montré que les moDC stimulées par les agrégats induisent la prolifération de lymphocytes TCD4+, dont la polarisation dépendait de la nature de la protéine. Ainsi les agrégats de GH conduisent à une production majoritaire d’IFNγ, signe d’une réponse de type Th1, tandis que les agrégats d’anticorps induisent une réponse mixte, Th1, Th2,Th17 (production d’IFNγ, IL-5, IL-13 et IL-17.Enfin, nous avons commencé l’étude des mécanismes intra cellulaires impliqués dans l’activation des moDC, en montrant que les agrégats de GH induisent la phosphorylation de p38MAPK, ERK, JNK et NF-κB (p65). Ces mêmes voies de signalisation sont impliquées dans l’expression de CXCL10,chimiokine connue pour induire la polarisation Th1.Au final, ces résultats confirment l’effet immunomodulateur des agrégats de protéines sur les cellules dendritiques et précisent leur rôle de signal de danger conduisant à la mise en place d’une réponse immunitaire contre les protéines thérapeutiques. / A major drawback in therapeutic biological products (BP) use is the development of anti-drug antibodies (ADA) in patients. Among other factors, BP aggregates seems to play a major role in immunogenicity. Moreover, the presence of ADA with high affinity and different isotypes suggest a CD4 T-cell dependent immune response and therefore a pivotal role for antigen presenting cells, such as dendritic dells (DC).In order to determine if BP aggregates participate to DC activation, aggregates form human growth hormone (GH) and antibodies (Rituximab and polyclonal IgG1) were produced and characterized.Their impact was tested on a model of monocytederived dendritic cells (mo-DC).We have shown aggregates were able to induce moDC maturation, as observed with increase of key co-stimulatory and maturation markers (CD40, CD80, CD83, CD86 and HLA-DR), and by increase of pro-inflammatory cytokines and chemokines (IL-6, IL-8, IL-12p40, CCL2, CCL3, CCL4, CXCL10).Using an allogenic model of co-culture, we have shown that moDC stimulated with aggregates were able to induce CD4+ T cells proliferation.Polarization was different following the nature of the protein. GH aggregates were able to induceIFNγ, sign of Th1 response, whereas antibody aggregates induced Th1, Th2, Th17 mix response (with production of IFNγ, IL-5, IL-13 and IL-17).Finally, we started to study intracellular mechanisms involved in moDC activation, by showing that GH aggregates were able to induce p38MAPK, ERK, JNK and NF-κB (p65)phosphorylation. These pathways are involved in CXCL10 expression, which is implicated in Th1 polarization. These results confirmed the immunomodulary effect of protein aggregates on DC and their role as danger signal, inducing an immune response against therapeutic proteins.

Les bactéries entéropathogènes du genre Salmonella et Shigella sont transmises par les aliments ou l’eau et sont responsables de nombreuses infections entériques chez les animaux et les humains. Ces maladies infectieuses restent une cause importante de morbidité et de mortalité dans les pays en voie de développement. L’existence de multiples sérotypes de Salmonella et de Shigella ainsi que l’émergence de souches résistantes aux antibiotiques, nécessite le développement de vaccins efficaces et large spectre. Ces bactéries utilisent un système d’injection de leurs protéines effectrices, appelé injectisome ou encore Système de Sécrétion de Type III (SST3), nécessaire à leur pathogénicité. Alors que les protéines effectrices injectées au moyen de cet injectisome sont variées et dépendent essentiellement du type cellulaire cible et donc de la spécificité du pathogène, certaines des protéines structurales composant l’injectisome sont relativement bien conservées parmi les différentes bactéries pathogènes, notamment les protéines de l’aiguille : PrgI et MxiH, et celles de la coiffe de l’aiguille : SipD et IpaD, respectivement de Salmonella et Shigella. Ces protéines, fortement impliquées dans la virulence des bactéries, semblent donc être des cibles de choix pour lutter contre des infections opportunistes impliquant ces bactéries pathogènes.Le premier objectif de cette thèse était d’évaluer l’immunogénicité et l’effet protecteur des protéines structurales de l’injectisome citées précédemment contre les infections à Salmonella et Shigella. Les protéines recombinantes préparées et produites au laboratoire ont été utilisées de façon séparée ou en combinaison pour immuniser des souris par différentes routes. Ensuite, les réponses immunitaires des souris ainsi immunisées ont été analysées par des tests immunométriques. Enfin, le potentiel immunogène et vaccinant de ces protéines structurales a été évalué en infectant les souris immunisées avec 100 DL50 de Salmonella par voie orale ou de Shigella par voie intranasale. Le meilleur résultat a été obtenu en utilisant la voie intra-gastrique pour les immunisations avec environ 70% de protection. Cette stratégie a permis également d’évaluer la pertinence de cette approche vaccinale dans un modèle murin de protection croisée (entre 25 et 60%). Le deuxième objectif de cette thèse était d’évaluer le pouvoir protecteur d’anticorps monoclonaux murins reconnaissant les régions conservées des protéines SipD et IpaD. Les anticorps obtenus ont été caractérisés et leur pouvoir neutralisant a été évalué in vivo dans un modèle murin d’infection avec Salmonella ou Shigella (jusqu’à 60% de protection).L’ensemble de ces travaux montre que l’utilisation de certaines protéines structurales conservées de l’injectisome de bactéries entéropathogènes présente un intérêt vaccinal et immunothérapeutique pour aider au traitement de certaines salmonelloses et shigelloses. / Salmonella and Shigella species are food and water borne pathogens that are responsible for enteric infections in both humans and animals. These infectious diseases are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of antibio- resistant strains, require the development of protective and broad-spectrum vaccines. All these bacteria utilize a system for injection of their effectors, called injectisome or Type III Secretion System (T3SS), necessary for their pathogenicity. While effector proteins are varied and depend essentially on the cellular target and thus on the specificity of the pathogen, the structural proteins that form the injectisome are common to all virulent Salmonella and Shigella spp., particularly the needle proteins PrgI and MxiH and the needle-tip proteins SipD and IpaD of Salmonella and Shigella respectively. These proteins, strongly involved in the virulence of the bacteria, appear to be ideal candidate antigens for a subunit-based, broad spectrum vaccine.The first aim of my PhD was to evaluate the immunogenicity and protective efficacy of structural proteins of the above-mentioned injectisome against Salmonella and Shigella infections. The recombinant proteins were prepared and produced in the laboratory and were used alone or in combination to immunize mice using different routes. The immune responses of immunized mice were then analyzed by immunometric assays. Finally, the protective efficacy was evaluated in a mouse model of intestinal (Salmonella) or pulmonary (Shigella) challenge. The best result was obtained by orogastric immunization with 70% of protection. This strategy also allowed to estimate the relevance of this approach in a mouse model of crossed protection (from 25 to 60%). The second objective of my PhD was to evaluate the protective efficacy of murine monoclonal antibodies recognizing conserved regions of SipD and IpaD proteins. The obtained antibodies were characterized and their therapeutic effect was evaluated in vivo with a Salmonella and Shigella infection murine model (up to 60% of protection).To conclude, this work showed that some conserved structural proteins composing the injectisome of enteropathogenic bacteria is of interest for treatment of enteric diseases caused by Salmonella and Shigella.

L’immunogénicité des biothérapies constitue une limitation majeure au traitement des patients atteints de maladies chroniques et se traduit par la production d’anticorps dirigés contre le biomédicament (anti-drug antibodies, ADA). La détection d’ADA de haute affinité et d’isotypes divers chez les patients suggère la mise en place d’une réponse immunitaire adaptative classique orchestrée par les cellules dendritiques. Par ailleurs, la présence d’agrégats protéiques dans les spécialités administrées serait un des facteurs favorisant l’immunogénicité, ces agrégats pouvant jouer le rôle de signal de danger.L’objectif de notre travail était de mieux comprendre les interactions des agrégats de protéines avec les cellules dendritiques et les lymphocytes T aboutissant au déclenchement d’une réponse immunitaire adaptative spécifique nécessaire à la production d’ADA.Dans un premier temps, nous avons montré que des agrégats d’infliximab, anticorps monoclonal anti-TNFa;, induisaient la maturation de cellules dendritiques humaines dérivées de monocytes (moDC). Celle-ci se traduit par l’augmentation de l’expression de marqueurs membranaires d’activation et de costimulation et la sécrétion de cytokines et chimiokines pro-inflammatoires. Nous avons également montré que ces modifications phénotypiques induites par les agrégats favorisaient la prolifération de lymphocytes T CD4+ et la production de cytokines. Par la suite, nous avons décrit les mécanismes cellulaires précoces impliqués dans l’activation de ces cellules en montrant que la neutralisation du récepteur FcgRIIa et de la tyrosine kinase Syk inhibait la maturation des moDC ainsi que l’activation des lymphocytes T CD4+.Par ailleurs, nous avons évalué le rôle des agrégats d’infliximab dans la génération de néo-épitopes, en mettant en évidence l’existence d’un répertoire de lymphocytes T CD4+ naïfs reconnaissant spécifiquement les agrégats d’infliximab, chez le sujet sain. Ainsi, grâce à des préparations d’agrégats bien caractérisées et d’un modèle de co-cultures autologues de lymphocytes T CD4+ naïfs et de moDC chargées avec les agrégats, nous avons montré que la fréquence de LT CD4+ naïfs spécifiques des agrégats d’infliximab est plus importante que celle retrouvée pour l’anticorps natif. Ces résultats suggèrent une présentation accrue d'épitopes et de néo-épitopes dérivant des agrégats d'infliximab.In fine, ce travail contribue à une meilleure compréhension des conséquences biologiques de l’agrégation des protéines à visée thérapeutique sur le déclenchement de la réponse immunitaire adaptative spécifique en mettant l’accent sur les rôles adjuvant et antigénique des agrégats protéiques. / Immunogenicity of biotherapeutic proteins is a major drawback in the treatment of patients with chronic diseases characterized by the production of anti-drug antibodies (ADA). The detection of ADA with high affinity and of various isotypes suggests a CD4 T cell-dependent adaptive immune response with a pivotal role for dendritic cells. Among other factors, the presence of protein aggregates in the administered products can promote immunogenicity, as aggregates seem to act as danger signals.The aim of our work is to better understand the interactions of protein aggregates with dendritic cells and T cells, leading to the establishment of a specific adaptive immune response needed for the production of ADA.We first showed that aggregation of infliximab, a monoclonal anti-TNFa; antibody, induced the maturation of human monocyte-derived dendritic cells (moDC) via an increase in the expression of activation and costimulatory surface markers and the secretion of pro-inflammatory cytokines and chemokines. Moreover, we showed that these phenotypic changes induced by aggregates promote CD4 T-cell proliferation and cytokine production. Subsequently, we described the early events involved in moDC and T-cell response by showing that the neutralization of the FcgRIIa receptor and the tyrosine kinase Syk inhibited moDC maturation and CD4 T-cell activation.Furthermore, we evaluated the involvement of infliximab aggregates in the generation of neo-epitopes by identifying a naïve CD4 T-cell repertoire recognizing infliximab aggregates in healthy subjects. By testing well characterized aggregate preparations and using an autologous co-culture model of naïve CD4 T cells and moDC loaded with aggregates, we showed that the frequency of naïve CD4 T cells specific for infliximab aggregates was higher than the one found for the native antibody. These results suggested an increased presentation of epitopes and neo-epitopes derived from infliximab aggregates.In resume, our work contributes to a better understanding of the biological consequences of therapeutic protein aggregation on the onset of the specific adaptive immune response by focusing on the adjuvant and antigenic role of protein aggregates.

This paper describes our continuing studies on stabilization of peptide structures in supersecondary conformations that are designed to mimic conformational antigenic epitopes. In this work we have used the consensus Cys2His2 zinc-finger peptide motif as a template to engineer and synthesize antigenic loop peptide segments from two protein antigens, lactate dehydrogenase C4 isozyme (LDH-C4) and human chorionic gonadotropin β subunit (β-hCG). Confirmation that the engineered peptide constructs assumed a zinc-finger conformation was obtained by absorption spectroscopy of the Co2+ complexes. The circular dichroism (CD) spectra of the free peptides show random coil conformations, while the Zn2+-complexed peptides acquired the zinc-finger motif upon titration with Zn2+, as evidenced by the appearance of absorbances indicating α-helix and some β-conformation. No peptide aggregation was observed, as these peptides were monomeric under all conditions tested. In order to examine the immunogenicity of the zinc-finger constructs, one sequence from LDH-C4 (ZFLMVF) and two sequences from β-hCG (ZF2TT3 and ZF4TT3) were selected and chimeras were synthesized to incorporate promiscuous T-cell epitopes from either tetanus toxoid or measles virus. The ZFLMVF construct was highly immunogenic in rabbits, and the ZF2TT3 and ZF4TT3 peptides were highly immunogenic in both mice and rabbits, eliciting high-titer antipeptide antibodies specific for their immunogenic sequences. However, the antibodies raised to the zinc-finger constructs showed minimal reactivity against their respective native protein antigens as determined by ELISA. This is surprising in the case of β-hCG, since the ZF2 zinc-finger peptide was an effective inhibitor of binding of anti-β-hCG-loop(38-57) antibodies to whole hCG, as assessed by a competitive inhibition radioimmunoassay. This implies that, although the cyclized 40-52 sequence from βhCG and the zinc-finger peptide ZF2 exhibit similar conformations in solution, the zinc-finger engineered loop is apparently not in a sufficiently correct conformation for antibody recognition of native hCG. Our results with the LDH-C4 zinc finger loop imply that antibody recognition of antigen involves specific side-chain interactions that must be maintained by a precise conformation.