Bakgrund: Malaria är en av de allvarligaste infektionssjukdomarna i världen. De allvarligaste malariafallen orsakas främst av Plasmodium falciparum som sprids av Anopheles-myggor. Ett vaccin med långvarigt och potent skydd skulle kunna minska dödligheten, men också minska behovet av kontrollåtgärder och problemet med läkemedelsresistens. Subenhetsvaccin är den vaccintyp som kommit längst i kliniska studier men dessa uppvisar begränsad effekt. Helparasitvaccin ger en bredare immunitet vilket kan ge ett mer fullständigt skydd. Syfte: Syftet med denna litteraturstudie var att jämföra effekt och varaktighet i skydd mot P. falciparum hos de två P. falciparum sporozoit (PfSPZ)-helparasitvaccinkandidaterna: RAS (Radiation-attenuated sporozoites) och CPS (Chemoprophylaxis and sporozoites), samt att undersöka betydelsen av vaccindos och administreringssätt. Metod: Arbetet är en litteraturstudie baserat på 14 vetenskapliga studier vilka har erhållits via sökning i PubMed. De aspekter som avhandlas är: vaccineffekt och dess varaktighet, immunsvar och dess korrelation till vaccineffekt, betydelsen av dos och administreringssätt samt vaccinens säkerhetsprofil. Resultat: Litteraturstudien visade att RAS-vaccin och CPS-vaccin kan ge ett potent samt säkert kort- och långvarigt skydd mot homolog kontrollerad human malaria-infektion (CHMI) vid immunisering via myggor eller venös inokulation. Dosen har stor betydelse för vaccineffekten och CPS-vaccin kan uppnå potent skydd vid mycket lägre doser än RAS-vaccin. En del immunmekanismer har visat sig korrelera med skydd men CD8+ T-celler i levern verkar ha störst betydelse för långvarigt sterilt skydd. Det långvariga skyddet mot heterolog kontra homolog CHMI är bristfälligt för både RAS-vaccin och CPS-vaccin. Slutsats: En potent vaccineffekt uppnås med PfSPZ-vaccin mot homolog CHMI vid tillräckligt hög dos, men inte ett långvarigt skydd mot heterolog CHMI vilket begränsar användningen i endemiska områden. / Background: Malaria is still one of the most common infectious diseases in the world and there is an overwhelming threat to the development of resistance to different control methods such as drugs and insecticides. A durable vaccine with sterile protection would reduce and maybe eradicate the disease. The most serious cases of malaria are caused by Plasmodium falciparum that is transmitted through the bites of infected Anopheles mosquitoes. The life cycle of malaria is extremely complex and different vaccine candidates have effects at different stages. Naturally acquired immunity develops gradually after many years of clinical episodes but never becomes sterile. RTS,S is the only vaccine candidate who has been in phase III clinical trials. Unfortunately this vaccine has limited efficacy, like many other subunit vaccines, due to rapidly diminishing antibody titers. Whole parasite vaccines have the ability to generate a greater quantity and breadth of antigenic exposure within both the humoral and cellular immunity. This results in stronger immune response and can provide sterile protection. The development of whole parasite vaccines has mainly focused on the pre-erythrocytic stage and the most tested vaccine candidates that are in early clinical trial are radiation-attenuated sporozoites (RAS), chemoprophylaxis and sporozoites (CPS) and genetically attenuated parasites (GAP). Aim: The purpose of this literature study is to examine and compare the vaccine efficacy and durability towards P. falciparum of the two whole parasite vaccine candidates: RAS and CPS and to examine the importance of dose and different routes of administration. Methods: Fourteen different clinical studies were selected from PubMed to be included in this literature study. Different variables were selected for study: the vaccine efficacy and it´s durability after controlled human malaria infection (CHMI) using P. falciparum parasites homologous or heterologous to the vaccine strain, the correlation between the immunogenicity and protection, the importance of the dose and different kinds of administration and vaccine safety. Results: According to the findings in the literature study, direct venous inoculation of RAS-vaccine and CPS-vaccine have the ability to give short and longlasting protection against CHMI using P. falciparum parasites homologous to the vaccine strain. The dose is of great importance to the vaccine efficacy and CPS-vaccine has the ability to give potent protection with much lower doses than RAS-vaccine. Some immune mechanisms in the blood correlate with protection but it seems to be the number of CD8+ T-cells in the liver that are of greatest importance for longlasting and steril protection. Whole parasite vaccines are safe but transient parasitemia is common when using CPS-vaccine. Unfortunately, vaccines with longlasting protection against CHMI using P. falciparum parasites heterologous to the vaccine strain has limited efficacy. Conclusion: RAS-vaccine and CPS-vaccine have the ability to give a potent vaccine efficacy against CHMI using P. falciparum parasites homologous to the vaccine strain when used in sufficiently high doses. Longterm protection against CHMI using P. falciparum parasites heterologous to the vaccine strain is limited and this in turn affects the use in endemic areas. In the future, the vaccine effect can be improved by higher doses, more infectious vaccine strains or vaccine cocktails. An alternative to RAS-vaccine and CPS-vaccine could be direct venous inoculation of late arresting GAP.

INTRODUÇÃO: A vacinação anual é recomendada como a medida mais efetiva contra a influenza sazonal. O Instituto Butantan (IB) realizou, anualmente, estudos clínicos das vacinas influenza sazonais trivalentes (fragmentada e inativada), produzidas em2013, 2014 e 2015. MÉTODO: Estudos de coorte prospectivos para descrever a imunogenicidade e a segurança da vacina influenza produzida pelo IB nos anos de 2013, 2014 e 2015 em participantes adultos saudáveis e idosos. Após assinatura do TCLE os participantes foram submetidos à coleta de sangue e receberam uma dose da vacina. Nos dias 1, 2 e 3 após a vacinação foram contatados para avaliação da segurança (reações adversas solicitadas locais e sistêmicas e não solicitadas). No dia 21(+7) pósvacinação retornaram ao centro de pesquisa para nova checagem da segurança e para a coleta de sangue para a avaliação da imunogenicidade pós-vacinação. As análises de imunogenicidade foram feitas através do método inibição de hemaglutinação (IH). Os desfechos de imunogenicidade foram: porcentagem de soroconversão (SC), porcentagem de soroproteção (SP) e razão da média geométrica dos títulos (RMGT) de anticorpos inibidores da hemaglutinação. O estudo de 2013 foi conduzido no Centro de Referência para Imunobiológicos Especiais (CRIE) e no Centro de Pesquisa Clínica do Instituto da Criança, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, os estudos de 2014 e 2015 foram realizados apenas no CRIE. As composições das vacinas utilizadas nos estudos em 2013, 2014 e 2015 seguiram as recomendações da OMS para vacina influenza sazonal do hemisfério sul. RESULTADOS: No ano de 2013, foram incluídos no estudo 47 adultos e 13 idosos, em 2014, 60 adultos e 60 idosos e em 2015, 62 adultos e 57 idosos. Nos estudos de 2013, 2014 e 2015, dor foi a reação adversa local mais frequente e cefaleia a sistêmica mais relatada. Todas as reações adversas observadas foram classificadas como leves ou moderadas e nenhuma como grave. Porcentagens de SP > 70% e >60% foram demonstradas para adultos e idosos, respectivamente, para os três vírus vacinais, nos estudos de 2013, 2014 e 2015. Porcentagem de SC > 40% foi demonstrada para os adultos, para os três vírus vacinais, apenas no estudo de 2014 e porcentagem de SC >30% foi demonstrada nos idosos, para os três vírus vacinais, apenas nos estudos de 2013 e 2014. RMGT > 2.5 nos adultos para os três vírus vacinais foi demonstrada apenas no estudo de 2013 e RMGT > 2 nos idosos, para os três vírus vacinais, foi demonstrada nos estudos de 2013, 2014 e 2015. CONCLUSÃO: As vacinas influenza sazonal de 2013, 2014 e 2015, produzidas integralmente pelo Instituto Butantan, foram seguras e imunogênicas segundo os parâmetros de imunogenicidade definidos pela EMA / INTRODUCTION: Annual vaccination is most effective way to prevent seasonal influenza illness. Instituto Butantan (IB) performed clinical studies with its 2013, 2014 and 2015 seasonal trivalent influenza vaccines (split-virion and inactivated) METHODS: Prospective cohort studies to describe the safety and immunogenicity of Instituto Butantan influenza vaccine, in healthy adults and elderly, from 2013 to 2015. Soon after the informed consent was signed, participants underwent blood collection followed by vaccination. On study days 1, 2 and 3 post vaccination participants were contacted by the stuffy to evaluate the occurrence of solicited (local and systemic) and non-solicited adverse reactions. On study day 21(+7) subjects returned to the clinical site for final safety assessments and blood collection for the immunogenicity evaluation post vaccination. The immunogenicity analyses were performed by means of haemagglutination inhibition assay (HI). The immunogenicity endpoints were: seroprotection (SPR) and seroconversion (SCR) rates and the geometric mean HI antibody titer ratio (GMTR). The 2013 study The Centro de Referência para Imunobiológicos Especiais (CRIE) and at the Centro de Pesquisa Clínica do Instituto da Criança, Hospital das Clínicas of Medical School of University of São Paulo and the 2014 and 2015 studies were conducted at CRIE. The vaccine composition followed the WHO recommendation for the southern hemisphere seasonal influenza vaccine RESULTS: 47 healthy adults and 13 elderly participated in the 2013 study, 60 healthy adults and 60 elderly in the 2014 study, and 62 healthy adults and 57 elderly in the 2015 study. In the 2013, 2014 and 2015 studies, pain was the most frequent local adverse reaction and headache the most frequent systemic adverse reaction. All observed adverse reactions were classified as mild or moderate and none as severe. SPR > 70% and SPR > 60% were observed in adults and elderly, respectively, for the three vaccine viruses, in the 2013, 2014 and 2015 studies. SCR > 40% was observed in adults, for the three vaccine viruses, only in the 2014 study and SCR > 30% was observed in elderly, for the three vaccine viruses, only in the 2013 and 2014 studies. GMTR >2.5 among adults, for the three vaccine viruses was only observed in the 2013 study and GMTR > 2.0 was observed among elderly, for the three vaccine viruses, in the 2013, 2014 and 2015 studies. CONCLUSION: The 2013, 2014 and 2015 seasonal influenza vaccines produced by Instituto Butantan were safe and immunogenic according to the immunogenicity criteria defined by EMA

INTRODUÇÃO: A vacinação anual é recomendada como a medida mais efetiva contra a influenza sazonal. O Instituto Butantan (IB) realizou, anualmente, estudos clínicos das vacinas influenza sazonais trivalentes (fragmentada e inativada), produzidas em2013, 2014 e 2015. MÉTODO: Estudos de coorte prospectivos para descrever a imunogenicidade e a segurança da vacina influenza produzida pelo IB nos anos de 2013, 2014 e 2015 em participantes adultos saudáveis e idosos. Após assinatura do TCLE os participantes foram submetidos à coleta de sangue e receberam uma dose da vacina. Nos dias 1, 2 e 3 após a vacinação foram contatados para avaliação da segurança (reações adversas solicitadas locais e sistêmicas e não solicitadas). No dia 21(+7) pósvacinação retornaram ao centro de pesquisa para nova checagem da segurança e para a coleta de sangue para a avaliação da imunogenicidade pós-vacinação. As análises de imunogenicidade foram feitas através do método inibição de hemaglutinação (IH). Os desfechos de imunogenicidade foram: porcentagem de soroconversão (SC), porcentagem de soroproteção (SP) e razão da média geométrica dos títulos (RMGT) de anticorpos inibidores da hemaglutinação. O estudo de 2013 foi conduzido no Centro de Referência para Imunobiológicos Especiais (CRIE) e no Centro de Pesquisa Clínica do Instituto da Criança, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, os estudos de 2014 e 2015 foram realizados apenas no CRIE. As composições das vacinas utilizadas nos estudos em 2013, 2014 e 2015 seguiram as recomendações da OMS para vacina influenza sazonal do hemisfério sul. RESULTADOS: No ano de 2013, foram incluídos no estudo 47 adultos e 13 idosos, em 2014, 60 adultos e 60 idosos e em 2015, 62 adultos e 57 idosos. Nos estudos de 2013, 2014 e 2015, dor foi a reação adversa local mais frequente e cefaleia a sistêmica mais relatada. Todas as reações adversas observadas foram classificadas como leves ou moderadas e nenhuma como grave. Porcentagens de SP > 70% e >60% foram demonstradas para adultos e idosos, respectivamente, para os três vírus vacinais, nos estudos de 2013, 2014 e 2015. Porcentagem de SC > 40% foi demonstrada para os adultos, para os três vírus vacinais, apenas no estudo de 2014 e porcentagem de SC >30% foi demonstrada nos idosos, para os três vírus vacinais, apenas nos estudos de 2013 e 2014. RMGT > 2.5 nos adultos para os três vírus vacinais foi demonstrada apenas no estudo de 2013 e RMGT > 2 nos idosos, para os três vírus vacinais, foi demonstrada nos estudos de 2013, 2014 e 2015. CONCLUSÃO: As vacinas influenza sazonal de 2013, 2014 e 2015, produzidas integralmente pelo Instituto Butantan, foram seguras e imunogênicas segundo os parâmetros de imunogenicidade definidos pela EMA / INTRODUCTION: Annual vaccination is most effective way to prevent seasonal influenza illness. Instituto Butantan (IB) performed clinical studies with its 2013, 2014 and 2015 seasonal trivalent influenza vaccines (split-virion and inactivated) METHODS: Prospective cohort studies to describe the safety and immunogenicity of Instituto Butantan influenza vaccine, in healthy adults and elderly, from 2013 to 2015. Soon after the informed consent was signed, participants underwent blood collection followed by vaccination. On study days 1, 2 and 3 post vaccination participants were contacted by the stuffy to evaluate the occurrence of solicited (local and systemic) and non-solicited adverse reactions. On study day 21(+7) subjects returned to the clinical site for final safety assessments and blood collection for the immunogenicity evaluation post vaccination. The immunogenicity analyses were performed by means of haemagglutination inhibition assay (HI). The immunogenicity endpoints were: seroprotection (SPR) and seroconversion (SCR) rates and the geometric mean HI antibody titer ratio (GMTR). The 2013 study The Centro de Referência para Imunobiológicos Especiais (CRIE) and at the Centro de Pesquisa Clínica do Instituto da Criança, Hospital das Clínicas of Medical School of University of São Paulo and the 2014 and 2015 studies were conducted at CRIE. The vaccine composition followed the WHO recommendation for the southern hemisphere seasonal influenza vaccine RESULTS: 47 healthy adults and 13 elderly participated in the 2013 study, 60 healthy adults and 60 elderly in the 2014 study, and 62 healthy adults and 57 elderly in the 2015 study. In the 2013, 2014 and 2015 studies, pain was the most frequent local adverse reaction and headache the most frequent systemic adverse reaction. All observed adverse reactions were classified as mild or moderate and none as severe. SPR > 70% and SPR > 60% were observed in adults and elderly, respectively, for the three vaccine viruses, in the 2013, 2014 and 2015 studies. SCR > 40% was observed in adults, for the three vaccine viruses, only in the 2014 study and SCR > 30% was observed in elderly, for the three vaccine viruses, only in the 2013 and 2014 studies. GMTR >2.5 among adults, for the three vaccine viruses was only observed in the 2013 study and GMTR > 2.0 was observed among elderly, for the three vaccine viruses, in the 2013, 2014 and 2015 studies. CONCLUSION: The 2013, 2014 and 2015 seasonal influenza vaccines produced by Instituto Butantan were safe and immunogenic according to the immunogenicity criteria defined by EMA

Le sujet de ce travail de thèse a concerné l'analyse de l’immunogénicité de progéniteurs cardiaques issus de cellules souches embryonnaires humaines. Le but a été double. D’une part, comprendre les mécanismes cellulaires et moléculaires qui sous-tendent cette immunogénicité et, d’autre part, mettre en place des stratégies d’immuno-intervention permettant de la surmonter.Le travail a comporté deux volets, l’un in vitro et l’autre in vivo utilisant des modèles expérimentaux murins. Les analyses in vitro, ont utilisé une méthode de culture lymphocytaire mixte où des progéniteurs cardiaques humains ont été mis en culture avec des lymphocytes allogéniques. Les résultats ont montré que les progéniteurs cardiaques sont effectivement immunogènes et que la réponse immunitaire qu’ils suscitent peut-être modulée efficacement par des cellules mésenchymateuses dérivées du tissu adipeux. De plus, nous avons confirmé l’expression des molécules d’histocompatibilité de classe I à la surface de progéniteurs cardiaques, une expression qui semble modulée au cours de la culture.Les modèles in vivo que nous avons utilisés ont consisté en l’implantation de corps embryoïdes et des progéniteurs cardiaques de souris dans un contexte allogénique. Divers sites d’implantation ont été utilisés (myocarde, capsule rénale, muscle gastrocnemius) chez des souris immunocompétentes. Les résultats ont montré qu’à la fois les corps embryoïdes et les progéniteurs cardiaques sont rejetés chez les receveurs immunocompétents non traités, avec une cinétique différente en fonction du site d’implantation. Par ailleurs, l’utilisation d’un traitement par anticorps anti-CD3, appliqué à différents temps suivant l’implantation nous a permis de prolonger la survie des cellules implantées en induisant, en fonction de la fenêtre thérapeutique, soit une immunosuppression soit une tolérance immunitaire. / The present work concerned the analysis of the immunogenicity of cardiac progenitors derived from human embryonic stem cells. Our aim was to understand the cellular and molecular mechanisms which underlie this immunogenicity and to surmount it by setting up strategies of immune-intervention. The study consisted in two major components, one in vitro and the other in vivo using experimental mice models. The in vitro analyses were assessed by the mixed leukocyte reaction method, where human cardiac progenitors were cultured with allogeneic lymphocytes. The results showed that the cardiac progenitors are indeed immunogenic and that the immune response that they induce could be modulated by mesenchymal stromal cells derived from adipose tissue. Moreover, we confirmed the expression of class I histocompatibility molecules on the surface of cardiac progenitors, an expression which seems modulated during the culture. The in vivo models that we used consisted of the grafting of embryoïdes bodies and cardiac progenitors derived from mouse embryonic stem cells in an allogeneic context. Cells were grafted in different sites of immunocompetent mice (myocardium, renal capsule, muscle gastrocnemius). The results showed highlighted that at the same time both embryoïdes bodies and cardiac progenitors are rejected among untreated immunocompetents hosts, whereas their survival is extended by anti-CD3 treatments, In addition, anti-CD3 treatment prolongs the survival of grafted cells, either by immunosuppression or by inducing immune tolerance according to the timing when it is applied.

Orientador: Marjorie de Assis Golim / Resumo: A infecção crônica pelo vírus da hepatite B (VHB) é a principal causa de cronificação da hepatite, cirrose hepática e carcinoma hepatocelular em humanos. A vacinação contra hepatite B é essencial a saúde da população, sendo a medida de menor custo e maior eficiência para controlar o vírus. A vacina desencadeia resposta imune com produção de anticorpos contra o antígeno de superfície do VHB (anti-HBs), contudo, alguns indivíduos não desenvolvem imunidade efetiva, havendo necessidade de doses adicionais. Assim, estimular a resposta imune nos indivíduos já vacinados, porém pouco respondedores ou não respondedores previamente, poderia contribuir para o aumento da produção de anticorpos e persistência dos mesmos ao longo do tempo. Considerando o potencial imunomodulador de β-glucanas, inclusive no aumento da ativação de células T e B em resposta a antígenos, propôs-se neste estudo avaliar a influência do uso de β-glucanas como suplemento alimentar em indivíduos com imunidade não efetiva pós-vacina, que necessitassem de dose booster. Foram incluídos 46 doadores de sangue do Hemocentro de Botucatu, com idade entre 18 anos e 25 anos completos, do sexo masculino, vacinados com três doses para hepatite B na primeira infância. Aqueles que apresentaram anti-HBs<10UI/L foram considerados não imunes, sendo mantidos no estudo (n=31) e divididos em dois grupos de forma aleatória. Ambos os grupos receberam booster da vacina, sendo um grupo suplementado via oral com cápsulas de amido (n=11; pl... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Chronic hepatitis B virus (HBV) infection is the main cause of hepatitis chronification, liver cirrhosis and hepatocellular carcinoma in humans. Vaccination against hepatitis B is essential to the health of the population, being the least costly and most efficient measure to control the virus. The vaccine triggers an immune response with the production of antibodies against HBV surface antigen (Anti-HBs), however, some individuals do not develop effective immunity, requiring additional doses. Thus, stimulating the immune response in individuals who have already been vaccinated, but with little or no previous response, could contribute to increased antibody production and persistence over time. Considering the immunomodulatory potential of β-glucans, including the increased activation of T and B cells in response to antigens, it was proposed in this study to evaluate the influence of the use of βglucans as a food supplement in individuals with post-vaccine ineffective immunity , who needed a booster dose. 46 blood donors from the Hemocenter of Botucatu, aged between 18 and 25 years old, male, who received three doses of hepatitis B vaccine in childhood were included. Those who had anti-HBs <10 IU / L were considered non-immune, being maintained in the study (n = 31) and randomly divided into two groups. Both groups received a vaccine booster, one group supplemented orally with starch capsules (n = 11; placebo - 500mg / day), and the other with β-glucans (n = 20; 500mg / day) f... (Complete abstract click electronic access below) / Mestre

Introducción: en la presente revisión conoceremos los detalles de esta nueva prueba de laboratorio, utilizada para cuantificar los anticuerpos neutralizantes contra el SARS-CoV-2. Esta prueba diagnóstica comienza a tener un mayor protagonismo, a razón del proceso de infección y vacunación en el mundo, para comprender los misterios del correlato de protección inmunológica. Contenido: los anticuerpos neutralizantes tienen la capacidad de bloquear la capacidad del virus, para unirse al receptor ACE2 en las células humanas y estos anticuerpos permiten eliminar el efecto de microorganismos invasores. Su actividad se genera por las proteínas situadas en la superficie de los virus, a las que se unen para «bloquear» la infección. Los anticuerpos neutralizantes se definen in vitro por su capacidad para bloquear la entrada, fusión o salida del coronavirus, es decir son anticuerpos funcionales. Resumen: en la actualidad existen diferentes pruebas de laboratorio (pruebas de inmunoensayo de alto rendimiento), que tienen la capacidad de detectar anticuerpos inmunoglobulinas G anti proteína S del SARS-CoV-2 y que se correlacionan con las pruebas de laboratorio gold standard para la determinación de estos anticuerpos. Es crucial que estas pruebas de inmunoensayo de alto rendimiento, sean validadas en su fabricación contra métodos gold standard para determinar la presencia de anticuerpos neutralizantes. Perspectiva: esta revisión pretende ampliar el conocimiento de esta nueva prueba, que en un futuro permitirán definir los valores de correlato inmunológico generados por las vacunas o por una infección previa.

Ce projet a accompagné le développement pré-clinique de l'hormone humaine Relaxine-2 (Rln2) ayant induit des anticorps durant des essais cliniques, il est axée autour de 2 problématiques : (1) comprendre son immunogénicité, (2) étudier l’impact de modifications chimiques sur l’immunogénicité afin d'augmenter sa stabilité.Compte tenu du rôle des lymphocytes T CD4 dans les réponses immunitaires, la fréquence de cellules T spécifiques de la Rln2 dans un large panel de donneurs sains a été estimée et a permis d’expliquer le développement d’anticorps anti-Rln2. La cartographie des épitopes T a ensuite identifié les zones portant son immunogénicité. Puis, 6 modifications chimiques (acide aminé D, acide aminé isobutyrique, peptoïde, N-méthylation, C-méthylation et réduction de la liaison peptidique) utilisées pour augmenter la demi-vie ont été introduites à la plupart des positions d’un peptide hautement immunogène. La reconnaissance par des cellules T, la liaison aux molécules de présentation et la capacité à induire des lymphocytes T CD4 ont été étudiées pour les peptides analogues modifiés. La plupart des modifications se sont révélées être très efficaces pour minimiser les propriétés immunogéniques.Ce projet de thèse se situe donc à la croisée des chemins entre l’acquisition de connaissances nouvelles en immunologie et leur application dans des processus de conception et de gestion des risques de peptides thérapeutiques. / This project has accompanied the pre-clinical development of the human hormone Relaxin-2 (Rln2) that induced antibodies during clinical trials, it focuses on two issues: (1) to understand its immunogenicity, (2) to study the impact of unnatural modifications on immunogenicity to increase its stability.Given the role of CD4 T-cells in immune responses, the frequency of Rln2-specific T-cells in a large panel of healthy donors was estimated, and explained the development of anti-Rln2 antibodies. The T epitope mapping then identified the areas responsible for its immunogenicity. Then, 6 unnatural modifications (D amino acid, amino isobutyric acid, peptoid, N-methylation, C-methylation & reduced peptide bond) used to increase the half-life were introduced at most positions of a highly immunogenic peptide. T-cell recognition, binding to HLA molecules and the ability to induce CD4 T-cells were studied for modified analog peptides. Most of the modifications were very effective in minimizing immunogenic properties.This thesis project is at the crossroads between the acquisition of new knowledge in immunology and its application in the process of design & risk management of therapeutic peptides.

La manipulation des anticorps monoclonaux thérapeutiques (Acm) reconstitués avant administration aux patients est susceptible d’entraîner leurs dégradations physiques (e.g. dénaturation, agrégation). Ceci peut avoir un impact sur leur efficacité et sécurité. Afin d’étudier la conformation et la stabilité des Acms reconstitués, nous avons développé des méthodes séparatives couplées avec la spectrométrie de masse (MS) en conditions non-dénaturantes (« native »). Une méthode de CZE-native MS utilisant un revêtement constitué d’une triple-couche ionique a été développée pour séparer et détecter les différentes conformations (monomère natifs, dénaturés, dimères) d’Infliximab. Une étude approfondie réalisée en analysant de l’infliximab digéré a permis d’établir que la formation du dimère était liée à la dénaturation du fragment Fab. L’intérêt des revêtements statiques (commerciaux et préparés in situ) ont été étudiés pour analyser les Acms par CZE-UV et CZE-MS. Nos résultats ont montré l’intérêt d’un nouveau revêtement monolithique. Un couplage simultané de la SEC avec la MS et un détecteur de fluorescence a été développé. Nous avons ainsi identifié les conditions expérimentales qui entraînaient des dénaturations et des dimérisations artificielles. Cette méthode a ensuite été également appliquée avec succès pour la caractérisation d’un échantillon de Trastuzumab stressé. Une méthode orthogonale en utilisant la SEC-mobilité ionique-MS a été employé pour évaluer la proportion de monomères dénaturés par rapport aux monomères natifs. La méthodologie ainsi développée permettra la détection de très faibles taux d'anticorps dégradés dans des poches d'infusion. Ceci permettra de définir les paramètres critiques à maîtriser lors de la reconstitution et la manipulation d’anticorps à usage hospitalier. / Manufacturing and manipulation of therapeutic monoclonal antibodies (mAb) in the hospital before administration to patient is prone to induce their physical degradations (e.g., denaturation, aggregation). This may impact their efficacy and safety. To study the stability of mAbs, capillary zone electrophoresis (CZE) and size exclusion chromatography (SEC), coupled to native mass spectrometry (MS) have been developed. CZE-native MS method using a triple-layer coating was developed to detect and separate different conformational states (unfolded monomer, dimer) of Infliximab in a single analysis. In-depth study with digested infliximab confirmed that dimer formation was related to the Fab fragment. We also focused on covalent coatings in order to find the more adapted coating to analyze mAbs by CZE-UV and CZE-MS. We also developed for SEC a simultaneous coupling with MS and a fluorescence detector to detect the degraded mAbs. We have identified the biases inducing conformational changes (e.g. dimerization, denaturation) that may arise during native MS. We also successfully characterized aggregates and denatured monomer in stressed Trastuzumab sample. In addition, the orthogonal method SEC-ion mobility-MS has been employed to separate and measure the denatured monomers compared to their related native conformations. Moreover, the developed system enables the detection of a very low levels of degraded mAbs in infusion bags. It allows to define the critical parameters to be controlled during the reconstitution and manipulation of therapeutic mAbs in hospital.

Bien qu’elle soit évitable par la vaccination, la grippe saisonnière est responsable annuellement de taux de morbidité et de mortalité élevés. Plusieurs études ont démontré que les facteurs sexuels (gènes et hormones) affectent la susceptibilité des individus aux maladies infectieuses et leur réponse aux vaccins. Toutefois, le sexe est souvent considéré comme une variable de confusion dans les études épidémiologiques, les résultats sont ainsi ajustés pour cette variable, ce qui rend la comparaison entre les deux sexes impossible. Le but de mon mémoire est de synthétiser les preuves existantes sur les différences liées au sexe dans la réponse au vaccin antigrippal. Ceci permettrait d’orienter les recommandations vaccinales qui tiendraient compte du sexe. Par conséquent, nous avons procédé à une revue systématique de la littérature afin d’analyser les données disponibles concernant les différences liées au sexe dans l’immunogénicité, l’efficacité potentielle, l’efficacité réelle et l’innocuité du vaccin contre la grippe saisonnière. Les résultats n’indiquent aucune différence dans l’immunogénicité et l’efficacité réelle du vaccin antigrippal saisonnier entre les sexes. Tandis que les taux de manifestations cliniques inhabituelles (MCI) étaient plus élevés chez les femmes. Enfin, il est nécessaire de disposer de données probantes afin de mieux comprendre les différences liées au sexe dans la réponse au vaccin antigrippal. / Every year, seasonal influenza is an important cause of morbidity and mortality, despite being vaccine-preventable. Several studies have demonstrated that sex factors (genes and hormones) impact individuals’ susceptibility and response to infectious diseases and vaccines. However, most studies do not explicitly assess sex differences in vaccine response despite collecting this data, but rather adjust for sex. The purpose of my dissertation is to synthesize the current evidence on sex differences in response to seasonal influenza vaccine in an attempt to guide sex-specific recommendations in influenza vaccines administration. Therefore, we conducted a systematic review to analyze available evidence on sex differences in immunogenicity, efficacy, effectiveness and/or safety of the seasonal influenza vaccine. Available data show no sex differences in the immunogenicity and effectiveness of seasonal influenza vaccine, while higher rates of adverse events following immunization (AEFIs) seem to occur in females. However, evidence of higher quality is needed to better understand sex differences in response to influenza vaccine.

Optimale Konservierungsmethoden sind erforderlich, um die bedarfsgerechte Verfügbarkeit kardiovaskulärer Transplantate für den Ersatz geschädigter Gewebe (Herzklappen oder Gefäße) zu garantieren. Die konventionelle Kryokonservierung (engl.: Conventional Frozen Cryopreservation, CFC) ist derzeit der Standard zur Konservierung kardiovaskulärer Allografts. Jedoch limitieren Immunreaktionen deren Langzeitfunktionalität. Die Alternative der eisfreien Kryokonservierung (engl.: Ice-free Cryopreservation, IFC) wurde kürzlich entwickelt. In der Arbeit wurde die Reaktion des humanen Immunsystems auf allogene kardiovaskuläre Gewebe nach Anwendung unterschiedlicher Konservierungsmethoden umfänglich charakterisiert. Zusätzlich wurde Glutaraldehyd (GA)-fixiertes Gewebe untersucht, um die Ergebnisse in den Gesamtkontext der Gewebekonservierung einzuordnen. Die Analyse des konservierten humanen Aortengewebes, welches als Modellmaterial diente, ergab, dass die Gewebestruktur nach IFC erhalten blieb, jedoch die metabolische Aktivität sowie Apoptose und Nekrose des Gewebes durch IFC reduziert wurde. Dies spiegelte sich auch in der verminderten Freisetzung von Zytokinen aus IFC-Gewebe wider. Funktionelle In-vitro-Tests zeigten deutlich, dass Immunzellen verstärkt in Richtung der löslichen Faktoren aus CFC- und GA-fixiertem Gewebe, jedoch nicht aus IFC-Gewebe migrieren. In Kokulturen der Makrophagen auf dem Aortengewebe konnte ausschließlich bei Makrophagen, welche auf GA-fixiertem Gewebe kultiviert wurden, eine Polarisation zum M1-Phänotyp festgestellt werden. Weiterhin zeigte sich, dass lediglich Faktoren des CFC-Gewebes in der Lage waren, die Aktivierung und Proliferation von T-Zellen zu verstärken. Insgesamt belegen diese Daten detailliert, dass IFC die Eigenschaften des Gewebes selektiv moduliert und dadurch eine verringerte Aktivierung des Immunsystems stattfindet. Die Ergebnisse verdeutlichen, dass IFC eine aussichtsreiche Strategie zur verbesserten Konservierung darstellt. / Optimal preservation methods are needed, to ensure constant availability of biological matrices for the replacement of damaged cardiovascular structures (heart valves or vessels). Conventional frozen cryopreservation (CFC) is currently the gold standard for cardiovascular allograft preservation. However, inflammation and structural deterioration limit transplant durability. The recently developed method of Ice-free cryopreservation (IFC) might be a superior method. The aim of this study was to characterize the reaction of the human immune system to allogeneic cardiovascular tissues after different cryopreservation methods. Regarding some aspects, the cryopreservation was compared to glutaraldehyde (GA) fixation, which is another common tissue preservation method. Human aortic tissue served as a proof-of-principle material for heart valves and vascular allografts. First, the histological and metabolic features of the differently preserved aortic tissues were analyzed. Tissues preserved by IFC exhibited typical architecture but significantly lower metabolic activity and the absence of necrotic or apoptotic cells. The reduced release of cytokines from IFC-tissue reflected these latter observations. In functional in-vitro-assays it was shown that migration of immune cells was significantly enhanced by soluble factors from CFC and GA-fixed tissue, but not by factors from IFC-tissue. In co-cultures of macrophages on aortic tissue, none of the preserved tissue induced activation. Exclusively GA-fixed tissue triggered the polarization of macrophages towards a M1-phenotype. Moreover, cues from only CFC-tissue but not IFC-tissue amplified T cell activation and proliferation. In conclusion, IFC selectively modulates the characteristics of tissues resulting in an attenuated activation of the human immune system. Therefore, IFC treatment is a promising strategy for improved tissue preservation and storage of cardiovascular allografts for clinical use.