Au cours des dernières décennies, la technologie des nanoparticules pour la délivrance des vaccins au niveau de muqueuses a reçu un intérêt croissant. L’administration intranasale possède de grands avantages pour la stimulation du système immunitaire, telles que la stimulation d’une immunité protectrice locale et systémique. Cependant des systèmes de délivrance et des adjuvants sont souvent nécessaires pour déclencher efficacement la réponse immunitaire. Nous avons appliqué la technologie des nanoparticules en tant que système de délivrance d'un vaccin universel nasal contre la grippe dans un projet européen FP7 appelé UniVacFlu. Nous avons formulé un antigène adjuvé CTA1-3M2e-DD avec les NPL. Cet antigène est composé de la sous-unité A1 de la toxine du choléra et d’un épitope conservé du virus de la grippe A (M2e), ainsi que du dimère de l’analogue synthétique de la protéine A de Staphylococcus aureus (DD). Les nanoparticules utilisées sont poreuses et constituées de maltodextrines réticulées ayant un coeur lipidique (NPL). L’association de cet antigène avec les NPL est quantitative et la formulation est stable pendant au moins six mois à 4°C. Les NPL permettent également de délivrer d’une manière accrue cet antigène dans les cellules épithéliales des voies respiratoires et les macrophages. Actuellement ces formulations sont évaluées chez la souris par le consortium UniVacFlu.L'un des principaux problèmes des vaccins nasal est la toxicité qui peut être provoquée par le passage nez-cerveau de l'un de ses composants. Le but de ce travail est d'évaluer le potentiel des NPL, en tant que vecteurs pour la délivrance des vaccins nasal. Ainsi, nous avons étudié le chargement d’un antigène dans les NPL et sa délivrance dans les cellules épithéliales des voies respiratoires. Notre étude révèle que les NPL interagissent fortement avec les muqueuses et délivrent d’une manière accrue les antigènes dans les cellules. Nous avons également montré l'absence de transcytose et de passage paracellulaire des NPL ou des antigènes délivrés dans un modèle de barrière épithéliale in vitro. Les résultats in vivo confirment l'absence de passage nez-cerveau des NPL et montrent qu’elles prolongent fortement le temps de résidence nasale des antigènes qui sont ensuite éliminés par le tractus gastro-intestinal.Ces résultats mettent en évidence l'intérêt des NPL comme vecteurs pour la prochaine génération de médicaments et de vaccins. / Nanoparticles technology for mucosal delivery of vaccines received a growing interest in the last decades. Intranasal administration owns great advantages for immune system stimulation, such as local and systemic protection against infectious diseases. However delivery systems and adjuvants are often required to efficiently trigger mucosal and systemic immune responses. In this thesis, nanoparticles (NP) have been evaluated as delivery system for a nasal universal influenza vaccine in a People Program of the European Union Seventh Framework Program FP7 called UniVacFlu. The aim of the UniVacFlu network is to develop a universal influenza vaccine administered through the mucosal route. We used porous maltodextrin nanoparticles with a lipidic core (NPL). We loaded an adjuvanted antigen named CTA1-3M2e-DD in the NPL. CTA1-3M2e-DD is composed of the A1 subunit of the cholera toxin and a conserved epitope of influenza A virus (M2e), while DD, dimer of the synthetic analogue of the Staphyloccous aureus protein A, targets B cells. Interestingly the antigen loading in NPL was quantitative for the antigen: NPL 1:5 mass ratio and the formulation was stable for at least six months at 4°C. We assessed the successful delivery of the antigen by NPL in airway epithelial cells and macrophages. These formulations are currently evaluated by the UniVacFlu consortium in mice.One of the main issues of intranasal vaccines is the toxicity that can be elicited by the nose-brain passage of one of their components. We investigated the loading of antigens in NPL and their delivery in airway mucosa. We observed a high endocytosis of NPL and an increased protein delivery into the cells. On a transwell model of the airway mucosa we assessed the absence of transcytosis and paracellular passage of the NPL. In vivo results confirmed the lack of nose-brain passage of the NPL, as NPL were found not to cross the mucosa. Interestingly, we observed an increased nasal residence time of the protein targeted by NPL. The particles after having delivered their payload are totally eliminated through the gastrointestinal tract, making these nanoparticles good candidates for mucosal delivery system. These results highlight the interest of NPL as vectors for mucosal delivery of drugs.

Vaccination comes second in importance after introduction of clean water as a public health intervention which has largely contributed in the reduction of deaths from infectious diseases. Success in the development of a group A streptococcal (GAS) vaccine is expected to save 517 000 deaths per annum according to a recent independent review commissioned by the world health organization (WHO) and would offer an ideal means to prevent rheumatic heart disease (responsible for the greatest health burden) and other GAS-associated diseases which affect the health of 600 million. Traditional vaccine approaches (killed or live attenuated) have demonstrated great success against many bacterial and viral infectious diseases, crowned by the global eradication of smallpox announce by the WHO in 1980 and near-to-be announced eradication of polio viral disease. However, application of traditional techniques in many cases such as HIV/AIDS, malaria, GAS and Mycobacteria tuberculosis, has not shown the same success. Risk associated with the use of live–attenuated pathogens, such as recurrence of virulence (e.g. HIV), development of autoimmune diseases (e.g. GAS), and difficulties of manufacture hindered the use of such approaches. Other vaccine approaches such as subunit vaccines (recombinant proteins) and carrier conjugated vaccine are also hindered by the lack of suitable adjuvants, carriers and delivery systems. The current thesis focused on the design, synthesis and evaluation of novel adjuvants and vaccine delivery systems against GAS. The first chapter reviews recent approaches in the field of GAS vaccine design and new findings in immunology which represent the basis of our novel strategies. The second chapter describes the design, synthesis and evaluation of a novel library of lipopeptides as self-adjuvanting GAS vaccine candidates, composed of: (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2d) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment, or incorporation of P25 and J14 into a lipid-core peptide system (LCP) on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side-chain. Mucosal surface of the nasal-oral route is a primary site of GAS infections. An ideal GAS vaccine would have to elicit both mucosal as well as systemic immune responses and hence would not only prevent the development of GAS-associated diseases but also would prevent primary GAS infections. Therefore, the nasal route is considered a highly promising route of vaccine administration to provide local as well as systemic immune responses against pathogens that utilize mucosal surface as site of infection. The third chapter includes immunological assessment of the lipopeptide vaccine library described in the second chapter following intranasal immunization of B10BR (H-2k) mice. The whole library was first investigated in a small scale experiment (5 mice per group) to select promising candidates which demonstrate the best local and systemic J14-specific antibodies. Four selected lipopeptides were further investigated in a larger scale experiment (15 mice per group) followed by intranasal challenge of vaccinated mice with a virulent GAS M1 strain. The best local and systemic immune responses were demonstrated by a lipopeptide featuring a lipid moiety consisting of two 16 carbon chains incorporated at the C-terminus of the lipopeptide. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide candidate featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side-chain. A possible explanation for these results was investigated where antibodies elicited by the former candidate was found to better recognize the minimal B-cell epitope in the native p145 sequence of the M protein. Circular dichroism study of lipopeptides used in the previous experiment demonstrated that the former candidate features α-helical conformation which is required to produce protective J14-specific antibodies. Further studies are needed to explain structural features required to achieve both α-helicity and strong mucosal immune responses shown by the previously mentioned two lipopeptides. Signaling through toll-like receptors expressed by immune cells was recently shown to result in a robust immune response and was investigated as a possible mode of action for our novel lipopeptides. The fourth chapter introduces our lipopeptide vaccine approach as novel synthetic ligands targeting TLR2. A lipid moiety consisting of two alkyl chains of 16 carbons was found to achieve optimal TLR2 signaling regardless of the position of lipid attachment. Carbohydrates as polyhydroxy compounds provide an easily accessible class of compounds to design scaffolds (carriers) to attach lipids and peptide epitopes in different number and stereochemical positions which makes glycolipopeptides an attractive target for adjuvant research and structure-adjuvanticity relationships studies. The Fifth chapter reports immunological assessment of two series of glycolipopeptides as GAS vaccine candidates and novel vaccine delivery systems. The first series: lipid carbohydrate core peptide system (LCCP); represents a modification of the classical LCP system where polylysine dendrimer is replaced by different monosccharides as carriers for peptide antigens. LCCP analogues induced proper humoral immune responses against incorporated epitopes comparable to the LCP delivery system and as strong as the immune response elicited by CFA mixtures. Moreover, LCCP delivery system has been proved to be tolerant to the use of different epitopes as well as changing carbohydrate cores. Design of novel carbohydrate cores with different orthogonal protecting groups is needed to explore the potential advantage of various stereochemical arrangements provided by monosaccharides. The second series of glycolipopeptides incorporates various glycolipid moieties (self-adjuvanting activity) covalently coupled to the N-terminus of J8 (a model epitope). The new glycolipopeptide vaccine candidates (containing only one copy of J8) bear comparison with an LCP analogue (containing four copies of J8) which would improve the ease of synthesis, purification and cost of vaccine production. The slight difference in immunogenicity among these glycolipopeptides was difficult to be explained due to intervening effects of both the number and orientation of lipids on immunological activity. Further investigation is needed to determine the contribution of each factor.

Vaccination comes second in importance after introduction of clean water as a public health intervention which has largely contributed in the reduction of deaths from infectious diseases. Success in the development of a group A streptococcal (GAS) vaccine is expected to save 517 000 deaths per annum according to a recent independent review commissioned by the world health organization (WHO) and would offer an ideal means to prevent rheumatic heart disease (responsible for the greatest health burden) and other GAS-associated diseases which affect the health of 600 million. Traditional vaccine approaches (killed or live attenuated) have demonstrated great success against many bacterial and viral infectious diseases, crowned by the global eradication of smallpox announce by the WHO in 1980 and near-to-be announced eradication of polio viral disease. However, application of traditional techniques in many cases such as HIV/AIDS, malaria, GAS and Mycobacteria tuberculosis, has not shown the same success. Risk associated with the use of live–attenuated pathogens, such as recurrence of virulence (e.g. HIV), development of autoimmune diseases (e.g. GAS), and difficulties of manufacture hindered the use of such approaches. Other vaccine approaches such as subunit vaccines (recombinant proteins) and carrier conjugated vaccine are also hindered by the lack of suitable adjuvants, carriers and delivery systems. The current thesis focused on the design, synthesis and evaluation of novel adjuvants and vaccine delivery systems against GAS. The first chapter reviews recent approaches in the field of GAS vaccine design and new findings in immunology which represent the basis of our novel strategies. The second chapter describes the design, synthesis and evaluation of a novel library of lipopeptides as self-adjuvanting GAS vaccine candidates, composed of: (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2d) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment, or incorporation of P25 and J14 into a lipid-core peptide system (LCP) on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side-chain. Mucosal surface of the nasal-oral route is a primary site of GAS infections. An ideal GAS vaccine would have to elicit both mucosal as well as systemic immune responses and hence would not only prevent the development of GAS-associated diseases but also would prevent primary GAS infections. Therefore, the nasal route is considered a highly promising route of vaccine administration to provide local as well as systemic immune responses against pathogens that utilize mucosal surface as site of infection. The third chapter includes immunological assessment of the lipopeptide vaccine library described in the second chapter following intranasal immunization of B10BR (H-2k) mice. The whole library was first investigated in a small scale experiment (5 mice per group) to select promising candidates which demonstrate the best local and systemic J14-specific antibodies. Four selected lipopeptides were further investigated in a larger scale experiment (15 mice per group) followed by intranasal challenge of vaccinated mice with a virulent GAS M1 strain. The best local and systemic immune responses were demonstrated by a lipopeptide featuring a lipid moiety consisting of two 16 carbon chains incorporated at the C-terminus of the lipopeptide. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide candidate featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side-chain. A possible explanation for these results was investigated where antibodies elicited by the former candidate was found to better recognize the minimal B-cell epitope in the native p145 sequence of the M protein. Circular dichroism study of lipopeptides used in the previous experiment demonstrated that the former candidate features α-helical conformation which is required to produce protective J14-specific antibodies. Further studies are needed to explain structural features required to achieve both α-helicity and strong mucosal immune responses shown by the previously mentioned two lipopeptides. Signaling through toll-like receptors expressed by immune cells was recently shown to result in a robust immune response and was investigated as a possible mode of action for our novel lipopeptides. The fourth chapter introduces our lipopeptide vaccine approach as novel synthetic ligands targeting TLR2. A lipid moiety consisting of two alkyl chains of 16 carbons was found to achieve optimal TLR2 signaling regardless of the position of lipid attachment. Carbohydrates as polyhydroxy compounds provide an easily accessible class of compounds to design scaffolds (carriers) to attach lipids and peptide epitopes in different number and stereochemical positions which makes glycolipopeptides an attractive target for adjuvant research and structure-adjuvanticity relationships studies. The Fifth chapter reports immunological assessment of two series of glycolipopeptides as GAS vaccine candidates and novel vaccine delivery systems. The first series: lipid carbohydrate core peptide system (LCCP); represents a modification of the classical LCP system where polylysine dendrimer is replaced by different monosccharides as carriers for peptide antigens. LCCP analogues induced proper humoral immune responses against incorporated epitopes comparable to the LCP delivery system and as strong as the immune response elicited by CFA mixtures. Moreover, LCCP delivery system has been proved to be tolerant to the use of different epitopes as well as changing carbohydrate cores. Design of novel carbohydrate cores with different orthogonal protecting groups is needed to explore the potential advantage of various stereochemical arrangements provided by monosaccharides. The second series of glycolipopeptides incorporates various glycolipid moieties (self-adjuvanting activity) covalently coupled to the N-terminus of J8 (a model epitope). The new glycolipopeptide vaccine candidates (containing only one copy of J8) bear comparison with an LCP analogue (containing four copies of J8) which would improve the ease of synthesis, purification and cost of vaccine production. The slight difference in immunogenicity among these glycolipopeptides was difficult to be explained due to intervening effects of both the number and orientation of lipids on immunological activity. Further investigation is needed to determine the contribution of each factor.

L'utilisation des nanobiotechnologies dans le domaine de la santé est en plein essor. Les nanoparticules de poly(acide lactique) (PLA) représentent un nanosystème biocompatible capable d'accroître la spécificité et l'efficacité de traitements thérapeutiques et vaccinaux administrables par voie muqueuse et intraveineuse. Toutefois, l'optimisation de ces nanosystèmes se heurte à une caractérisation incomplète de leur biodistribution in vivo, en particulier à l'échelle cellulaire.L'objectif de ce travail de thèse est d'enrichir les connaissances sur la biodistribution des nanoparticules de PLA in vivo après administration muqueuse ou intraveineuse, dans le but d'élargir les perspectives d'optimisation et d'utilisation. Animal complexe et adapté pour les études sur organisme-entier, le modèle du poisson-zèbre (Danio rerio) a été utilisé. Pour mener à bien ce projet, une méthodologie rigoureuse d'analyse de la biodistribution des nanoparticules de PLA a été développée. Ce qui permit, après administration par balnéation, d'en révéler le fort tropisme inné envers les cellules dendritiques muqueuses. Ces données ont servi à élaborer une stratégie de ciblage, utilisant la lectine agglutinine de cacahuète, capable d'augmenter la prise en charge des nanoparticules de PLA par les branchies et la peau. Enfin, l'étude du devenir de ces nanoparticules après injection intraveineuse, a révélé de nombreuses interactions avec le système circulatoire. Ce travail a permis d'approfondir la connaissance des interactions des nanoparticules de PLA avec le vivant, soulignant le potentiel prometteur de ces nanoparticules pour la vaccination muqueuse / Medecine shows a growing interest regarding nanobiotechnologies. Among them are poly(lactic acid) (PLA) nanoparticles, which represent a biocompatible and competent nanosystem to heighten the specificity and efficacy of diverse therapeutic and vaccine treatments, following mucosal and intravenous administration. However, the further optimization of such nanosystem is poised by the lack of informations regarding their in vivo biodistribution, especially at the cellular level.The main objective of this PhD is to increment the knowledge about PLA nanoparticles biodistribution in vivo, after muquous and intravenous administration, to further expand their optimisation and use perspectives. The zebrafish model has been utilized to perform this research because of his conserved complexity as well as his suitability for whole-organism studies.To fulfill this project, a precise methodology has been developed to analyze the PLA nanoparticles biodistribution. Which allowed, after bathing administriation, to unveil their robust innate tropism toward mucous dendritic cells. From these data has been established a targeting strategy, utilizing the peanut agglutinin lectin, which has been proved to enhance nanoparticle uptakes by both gills and skin mucosae. Finally, the study of PLA nanoparticles behavior and destiny after intravenous injection, revealed numerous elaborated interactions with the circulatory system.Overall, this work has been able to strengthen our understandings of PLA nanoparticles among living organisms, furthermore highlighting their promizing potential as nanovehicles for mucosal vaccines