The design and discovery of new surface-active polymers that self-assemble on solid substrates to form brush layers will have a major impact on numerous applications. Through recombinant DNA technology, there exists the potential to harness a cell's protein synthesis machinery to produce a brush-forming poly(amino acid) (or PAA) with an exactly specified amino acid sequence, thus controlling the polymer's composition at a level unequaled by conventional organic polymer synthesis. The presented work demonstrates the cloning, expression, purification and characterization of de novo-designed PAA's designed to form brush layers on alumina surfaces. Using conventional recombinant DNA methods, the feasibility of producing a PAA consisting of a poly-glutamate block and a poly-proline block was demonstrated. However, the PAA design was limited by the inherent limitations of conventional cloning techniques. We introduce here the development of a simple and versatile strategy for producing de novo-designed, high molecular weight PAA's using recombinant DNA technology. The basis of this strategy is that small DNA modules encoding for short PAA blocks can be easily inserted directly into a commercially available and unmodified expression vector. The insertions can be made repeatedly until the gene encodes for a polymer of desired molecular weight and composition. Thus, sequential modifications can be made to the PAA without having to re-start the gene assembly process from the beginning, thereby allowing for quick determination of how these changes affect polymer structure and function. The feasibility and simplicity of this method was shown during the production of a PAA, consisting of a long zwitterionic tail block and a short acidic anchor block, designed to form optimal brush layers on alumina surfaces. The success and flexibility of this method indicates that it can be applied for production of de novo-designed polypeptides in general. It is hoped that this method will contribute towards the rapid development of bio-inspired protein-based polymers for a variety of applications. This dissertation also contains research that aimed to use phage display technology to develop a new liposome-based immunoassay against biological toxins. This work was part of a collaboration effort with the U.S. Department of Defense and Luna Innovations. / Ph. D.

Neospora caninum is an apicomplexan parasite that causes neuromuscular paralysis in dogs and abortions in cattle. N. caninum is responsible for losses of several million dollars to the dairy and beef industries in several parts of the world. The key players in the host immune response to N. caninum include CD4+ T cells, the Th1 cytokines IL-12, Interferon gamma and IgG2a isotype antibodies. There are currently no chemotherapeutic agents that are effective against adult cattle neosporosis. A commercially available, inactivated vaccine induces the undesirable Th2 type of immunity against N. caninum. Therefore, two approaches towards vaccine development against N. caninum that were designed to induce potent cell mediated immunity have been explored in this dissertation. The first approach consisted of the development of a bivalent recombinant vaccine for both brucellosis and neosporosis, while the second approach involved gamma irradiation of N. caninum tachyzoites for use as an attenuated vaccine against N. caninum. Since N. caninum research has been conducted with several strains of mice and the different strains of mice vary in their susceptibility to infection with N. caninum, there is a need to develop a standard lab animal model for N. caninum. A gerbil and a C57BL/6 mouse model for N. caninum vaccine testing have been developed. It was found that the LD50 of N. caninum tachyzoites in gerbils was 9.3 x105 tachyzoites per gerbil delivered intra-peritoneally, (i.p) while for C57BL/6 mice the LD50 was 1.5 x107 tachyzoites per mouse delivered i.p. Vertical transmission rates in C57BL/6 mice infected with N. caninum tachyzoites during mid-gestation were determined and found to be in the range of 96-100%. Putative protective antigens of N. caninum that included MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in B. abortus strain RB51 to create recombinant vaccine strains. C57BL/6 mice were vaccinated with either the recombinant strains or the irradiated tachyzoites. Antigen specific IgG2a and IgG1 responses and high levels of interferon gamma and IL-10 were induced by vaccination. Mice vaccinated with irradiated tachyzoites, RB51-MIC1 and RB51-GRA6 were completely protected against lethal challenge, while the mice vaccinated with RB51-SRS2, RB51-GRA2 and RB51-MIC3 were partially protected. To determine the efficacy of the vaccines in preventing vertical transmission of N. caninum, mice were vaccinated and bred after administration of a booster dose four weeks after the primary vaccination. Antigen specific IgG1 and IgG2a and significant levels of IFN-ã and IL-10 were detected in vaccinated, pregnant mice. Pregnant mice were challenged with 5 x 106 N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups three weeks after birth and examined for the presence of N. caninum by a semi-nested PCR. Protection against vertical transmission elicited by the RB51-GRA6, RB51-MIC3, irradiated tachyzoite, RB51-GRA2, RB51-MIC1 and RB51-SRS2 vaccinated groups were 43%, 38%, 34%, 34%, 18%, and 7% respectively. Since not all the antigens that were highly protective against acute disease were not very effective in preventing vertical transmission, the role of the selected antigens in preventing acute disease and vertical transmission appear to differ. Only GRA6 was found to be effective in protecting against an acute lethal challenge as well as preventing vertical transmission 43% of the time. In summary, two animal models for the testing of N. caninum vaccines were developed. N. caninum protective antigens were successfully expressed in B. abortus strain RB51. The irradiated tachyzoite and recombinant RB51-Neospora vaccines were highly effective in protecting against acute neosporosis and partially protective against vertical transmission. Therefore, both these approaches show great promise as practical and effective means to achieve the goal of successful prophylaxis against N. caninum induced abortions and reduce the chances of vertical transmission. / Ph. D.

Aspects of recombinant enzyme production were characterized in genetically engineered Escherichia coli. Five strains transformed with the xylose isomerase overproduction system (pRK248/pTXI-1) were compared, based on parameters of cell metabolism and inducible enzyme activity in shake flask cultures. E. coli strain LE392 (pRK248/pTXI-l) performed the best with respect to nearly all of the parameters tested. The stability of the overproducing plasmids was tested in prolonged serial shake flask cultures. Segregational instability occurred in cultures lacking antibiotic selective pressure. The onset of this instability was influenced by host strain, plasmid construct and cultivation media. Despite its occurrence, the degree of plasmid instability, even in the worst case, would not be detrimental in the proposed application of this overproduction system. The cell mass and enzyme production of strain LE392 (pRK248/pTXI-1) was characterized in four batch cultures. It was determined that both carbon source starvation and the accumulation of acetate as a byproduct of glucose metabolism diminished the E. coli's ability to produce xylose isomerase. The peak enzyme production was determined to be 1320 International Units (IU) of xylose isomerase activity per liter of culture. The cell mass and enzyme production of strain LE392 (pRK248/pTXI-l) was studied in fed-batch cultures. The amount of xylose isomerase the cells produced was dependent on the degree of glucose limitation they experienced during cultivation. The peak enzyme production was 3250 IU/L. The results of these experiments were interpreted in the context of this overproduction system and its proposed application for the production of ethanol from plant biomass. / Ph. D.

Trichomonas vaginalis is a human urogenital parasite and the causal agent of trichomoniasis, the most common non-viral, sexually transmitted disease in the United States. Much of the pathogenic properties of T. Vaginalis stem from cysteine proteases. Here, we present the results of several studies on one variant, TvCP2, including purification and characterization of its active form, gene regulation in response to iron and oxygen, and localization and trafficking. Homologous to human Cathepsin L, TvCP2 was hypothesized to function as a protease, presumably localize to lysosomes, and play a role in T. vaginalis pathogenesis that is distinct from TvCP4. Levels of bacterially-expressed TvCP2 decreased faster in activation assays of lower pH. In Pichia pastoris, the amounts and form of TvCP2 expressed were variable, and protease activity was influenced by reducing agent and pH. Post-translational modifications may be in effect, or TvCP2 may be autocatalytic, however, actual autocatalytic processing remains to be determined. Consistent with previous reports, and contrary to TvCP4 regulation, TvCP2 mRNA levels were increased in T. vaginalis grown in media with reduced iron supplementation. Expression of processed TvCP2 protein increased, demonstrating post-transcriptional regulation and the potential for iron to influence processing of and/or proper sorting of TvCP2. Also, unlike TvCP4 expresion, which is unaffected by oxygen, both TvCP2 protein and mRNA were greatest under anaerobic conditions, suggestion transcriptional and translational regulation by oxygen, and that upon initial infection TvCP2 is not required immediately. Although overall immunofluorescent staining patterns were different between TvCP2 and TvCP4, hinting at distinct functions, both localized bto punctate vesicles, for which some colocalization was observed. Additionally, unlike TvCP4, TvCP2 did not colocalize with Vamp1/2 and did colocalize with legumain. These data suggest that TvCP2 is intracellular, targeted to lysosomes, and sorted independently from TvCP4. In conclusion, TvCP2 may play a unique role in the cell and is important for the life cycle and pathogenesis of T. vaginalis.

Yes / Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been contro- versial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that sev- eral of these specifically recognize EPOR by Western blot, immunoprecipi- tation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment. / Study was supported by the FP7-Health European commission EpoCan grant (282551).

Cette thèse de doctorat présente 3 études reliées à l’hybridation d’ADN fixée à un support solide et les phénomènes qui y sont associés. L’hybridation d’ADN en phase aqueuse est bien caractérisée, cependant plusieurs phénomènes et paramètres restent encore à déterminer lorsqu’un des brins est fixé sur un support solide. Un de ces paramètres est l’influence du brin complémentaire en fonction de la longueur de l’extrémité d’ADN cible immobilisée exposée à la phase aqueuse. Pour mieux comprendre ce phénomène, nous avons établi une méthode pour digérer spécifiquement le brin d’ADN complémentaire pour simplifier l’hybridation sur support solide. La combinaison d’une molécule favorisant la digestion du brin complémentaire et d’une molécule bloquant la digestion du brin cible a permis de produire un ADN cible simple brin à l’aide de l’exonucléase Lambda, permettant d’obtenir des signaux d’hybridation supérieurs à ceux obtenus avec des ADN doubles brins. Ensuite, l’observation en temps réel de l’hybridation d’amplicons simples brins et doubles brins a permis de mieux comprendre les phénomènes de compétition entre le brin complémentaire et la cible. Finalement, les conclusions des deux premières études nous ont permis d’inventer une technologie tirant profit de la compétition entre le brin complémentaire et la sonde de capture et de l’activité exonucléase pour réaliser l’amplification PCR et l’hybridation sur biopuce en un seul site réactionnel et dans un seul tampon réactionnel. La combinaison de ces techniques aura pour effet une simplification des dispositifs et une diminution du nombre de réactifs permettant la fabrication de tests diagnostiques automatisés à moindre coûts. Les travaux de cette thèse ont permis d’approfondir nos connaissances sur les phénomènes reliés à l’hybridation sur support solide et de concevoir une technologie faisant l’objet d’une demande de brevet. La suite logique de ces travaux serait de poursuivre le développement et l’optimisation de la technologie pour augmenter ses capacités de multiplexage tout en l’intégrant à un dispositif microfluidique et à un instrument de détection qui combine amplification PCR et hybridation sur biopuce dans une seule réaction réalisée en une seule étape. / This thesis presents three studies related to the phenomenon related to DNA hybridization onto capture probes attached to solid support. The work was carried out in the context of infectious diseases detection, but the conclusions are of interest to any test based on the recognition of genetic material by solid support immobilized capture probes. DNA hybridization in aqueous phase is well characterised, however several parameters and phenomena related to hybridization onto solid supports are still to be determined. One of these parameters is the influence of the complementary strand in relation with the length of the solvent-exposed immobilized target strand. To better understand this phenomenon, we established a method to specifically digest the complementary DNA strands to simplify hybridization onto solid support. The presence of a molecule favorising digestion on the complementary strand and a blocking molecule on the target strand enabled selective digestion of the complementary strand using Lambda exonuclease in PCR buffer. Hybridization of single-stranded target DNA generated this way resulted in significantly higher fluorescence signals than those obtained with double stranded hybridization. Afterwards, observation of real-time hybridization of single-stranded amplicons and double-stranded amplicons to capture probes fixed onto solid support allowed for a better understanding of the competition phenomena between the complementary strand and the capture probe. Finally, the knowledge gained with the first two studies was used to invent a technology taking advantage of the competition between the complementary strand and the capture probe and exonuclease activity to perform single vessel and single buffer PCR amplification and microarray hybridization. The combination of these techniques will simplify devices and require fewer reagents storage and handling, facilitating production of automated diagnostic tests at lower costs. The works of this thesis have expended our understanding of the phenomena associated with hybridization onto a solid support and also resulted in the creation of a new technology covered by a patent application. The logical progression of this work is to further develop and optimize this technology by integrating it into a microfluidic device and an instrument compatible with the detection of microarrays to increase the multiplexing capabilities of molecular diagnostics.

Thesis (MScEng)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Recombinant glycoproteins are important biopharmaceuticals, providing solutions for numerous previously untreatable illnesses, in everything from cancer to infertility. Most recombinant biopharmaceuticals are produced in mammalian cells due to their ability to provide the correct post-translational processing for use in humans. The post-translation processing influences many of the protein’s properties including pharmacokinetics, bioactivity, secretion, half-life, solubility, recognition and antigenicity. The aim of this thesis is to further study the upstream production of a glycosylated recombinant protein produced by Chinese hamster ovary (CHO) cells on production scale within the confines of an existing process. The process in question uses adherent CHO cells to produce a glycosylated recombinant hormone. As with most recombinant protein production processes, this process has two sections to the upstream production: a seed train to grow enough cells to inoculate production, and a production section, which focuses on the production of a recombinant protein. The seed train is predominantly conducted in roller bottles, while the production section takes place in perfusion bioreactors, where the cells are attached to microcarriers, with spin-filters for cell retention. The whole process uses medium with serum. There are two process challenges regarding an existing recombinant-protein production process: 1. The gradual increase, over the past several campaigns, of the final population doubling level of the cells (which must remain within certain specified limits) at the end of the seed train. 2. The low glycosylation levels of the product seen in certain campaigns, which meant that a certain number of final product batches were below the specified acceptable glycosylation limits. Following a literature survey several controlled process variables were chosen for investigation and hypotheses made on their effect on the seed train or glycosylation. To investigate their effect on the PDL and cell growth in the seed train: - Medium volume: decreasing the medium volume will yield a lower PDL due to slower cell growth caused by lower glucose availability. - Seeding density: if cells obtain confluence by the time they are harvested, decreasing the seeding density will yield a higher PDL. - Cultivation temperature: decreasing the temperature ought to decrease the growth rate. - Medium feed temperature: there will be no significant difference to the cell culture when pre-heated or cold medium is used. Aeration: using vent caps will increase the oxygen content of the medium in the roller bottles and the cell growth, yielding a higher PDL. To investigate their effect on glycosylation during production: - pH: better glycosylation will be seen at pH 6.9, than at pH 6.7. - Perfusion rate: a higher perfusion rate will lead to better glycosylation due to increased glucose and glutamine concentrations. In the seed train, the only factor that significantly influenced the final PDL was the seeding density. Cell growth was inhibited once cells reached confluence, so lowering the seeding density lead to a higher PDL. It is recommended to use a high seeding density to ensure a lower PDL. Historic data indicated that the seeding density was not the cause of the apparent increase of the final PDL, as all previous campaigns had been seeded with a high seeding density. What then became apparent was that the final PDL remained relatively constant during a campaign and that the increase in final PDL occurred between campaigns. It appears that the apparent increase in the final PDL is due to differences in cell counting between operators as each new campaign was managed by different operators. It is recommended that a mechanical cell counter be used to verify cells counts and to maintain a standard between campaigns. In the bioreactors, varying the pH proved to have no significant effect on the glycosylation levels. However, both the initial perfusion rate and the specific perfusion rate proved to be important from both historical data and the data generated during these experiments. Lower levels of the initial perfusion rate lead to better glycosylation and it is recommended that an initial perfusion rate of 1.0 volumes/day be used. The relationship between the specific perfusion rate and the glycosylation appears to be non-linear and requires further study, for now it is recommended that the specific perfusion rate be kept below 0.3 volumes/day/109 cells. Probable reasons for the unsatisfactory glycosylation seen in certain runs could also be proposed from these two factors: • RP33-133 : Very high specific perfusion rate • RP32-135 : High initial perfusion rate and very high specific perfusion rate • RP32-138 : High initial perfusion rate • RP33-139 : High initial perfusion rate Further research is recommended into the effect of the specific perfusion rate as well as the specific glucose consumption rate and the specific glutamine concentration on the glycosylation. / AFRIKAANSE OPSOMMING: Rekombinante glikoproteïene is baie belangrike biofarmaseutiese produkte wat oplossings bied vir talle voorheen ongeneeslike siektes in alles van kanker tot onvrugbaarheid. Meeste rekombinante farmaseutiese produkte word gemaak deur diere-selle as gevolg van hulle bevoegtheid om die korrekte na-translasie stappe te volg sodat die produkte in mense gebruik kan word. Die na-translasie stappe beïnvloed baie van die proteïene se karaktertreke insluitende die farmakokinetika, bioaktiwiteit, uitskeiding, half-leeftyd, oplosbaarheid, herkenbaarheid and antigeniciteit. Die doel van hierdie tesis is om die stroomop produksie van ‘n rekombinante glikoproteïene vervaardig deur Chinese hamster ovariale (CHO) selle verder te bestudeer binne die grense van ‘n bestaande proses op grootskaalse vlak. Die huidige proses gebruik CHO selle om ‘n rekombinante glikohormoon te produseer. Soos meeste prosesse wat rekombinante proteïene produseer bestaan die stroomop gedeelte van die proses uit twee dele: ‘n saad trein wat genoeg selle maak vir produksie en ‘n produksie gedeelte wat fokus op die vervaardiging van die glikoproteïen. Die saad trein bestaan hoofsaaklik uit roller bottels terwyl produksie plaasvind in perfusie bioreaktors waar die selle op “microcarriers” groei, met spin-filters om die selle binne die bioreaktors te hou; die hele proses gebruik medium met serum. Daar is twee probleme in die stroomop gedeelte van die bestaande proses: 1. Die geleidelike toename oor die afgelope paar jaar van die finale verdubbelingsvlak van die selle aan die einde van die saad trein 2. Die lae glukosilering van die eindproduk wat veroorsaak dat sekere lotnommers buite spesifikasie is Na ‘n literatuur studie, was seker beheerde proses parameters gekies om verder te bestudeer en hipotesisse gemaak oor hulle effek op die saad trein of die vlak van glukosilering. Die volgende faktore is bestudeer vir hulle effek op die finale verdubbelingsvlak van die selle in die saad trein: - Medium volume: ‘n laer medium volume sal lei tot a laer verdubbelingsvlak van die selle as gevolg van stadige groei - Konsentrasie van selle vir inokulasie: as die selle konfluent is teen die tyd wat hulle versamel word sal ‘n laer konsentrasie selle lei tot ’n hoër verdubellingsvlak. - Temperatuur: laer temperatuur behoort te lei tot ‘n stadiger groei koers van die selle - Medium voer-temperatuur: die voer-temperatuur van die medium sal geen beduidende verskil maak - Belugting: die gebruik van “vent-caps” sal die suurstof inhoud van die roller bottels verhoog Die volgende faktore is bestudeer vir hulle effek op die glukosilering tydens produksie: - pH: beter glukosilering word verwag by by pH 6.9 dan by pH 6.7 - Perfusie koers: ‘n hoër perfusie koers sal lei tot beter glukosilering as gevolg van hoër glukose en glutamien konsentrasies Die konsentrasie van die selle wat gebruik word vir inokulasie blyk die enigste faktor te wees wat die finale verdubbelingsvlak van die selle en die groei van die selle in die saad trein beïnvloed het. Die groei van die selle was beprek wanneer die selle konfluent geraak het en dus het ‘n laër sel konsentrasie by inokulasie gelei tot ‘n hoër sel verdubbelingsvlak. Dit word aanbeveel dat ‘n hoë sel konsentrasie by inokulasie gebruik word. Die geleidelike toename van die finale verdubbelingsvlak van die selle in die saad trein is waarskynlik as gevolg van die variasie in sel tellings tussen verskillende operateurs eerder as as gevolg van die beheerde proses parameters. Dit word aanbeveel dat ‘n meganiese sel-teller gebruik word om die verskil in sel tellings tussen operateurs te kontroleer en om ‘n standaard te handhaaf tussen produksie lotte. In die bioreaktors, het die pH geen beduidende invloed gehad op die glukosilering maar uit historiese data en die huidige data van hierdie eksperimente blyk albei die begin perfusie koers en die spesifieke perfusie koers ‘n belangrike invloed te hê op die glukosilering. Laër vlakke van die begin perfusie koers lei tot beter glikosilsie en dit word aanbeveel dat elke produksielot ‘n begin perfusie koers het van 1.0 volume/dag. Die verhouding tussen die glukosilering en die spesifieke perfusie koers blyk om nie-liniêr te wees nie. Nog navorsing hieroor word aanbeveel, maar vir nou word dit aanbeveel dat die spesifieke perfusie koers onder 0.3 volumes/dag/109 selle gehou word. Hierde twee faktore blyk die oorsaak te wees vir die lae glukosilering wat in sekere produksielopies gevind was: • RP33-133 : baie hoë spesifieke perfusie koers • RP32-135 : hoë begin perfusie koers en baie hoe spesifieke perfusie koers • RP32-138 : hoë begin perfusie koers • RP33-139 : hoë begin perfusie koers Dit word aanbeveel dat verdere navorsing gedoen word op die effek van die spesifieke perfusie koers asook die spesifieke koers van glukose verbruik en die spesifieke glutamien konsentrasie op die glukosilering van die produk.

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Les cellules dendritiques (CD) jouent un rôle majeur dans l'initiation, la régulation et le maintien des réponses immunes contre les pathogènes. Le ciblage d'antigènes (Ag) liés à des anticorps monoclonaux (AcM) dirigés contre des récepteurs spécifiques de CD est une approche vaccinale prometteuse induisant une réponse immunitaire chez l'animal. Cependant, certaines protéines de fusion AcM-Ag ne peuvent pas être produites. J'ai donc développé des AcM fusionnés à un domaine dockérine et des Ag fusionnés à un domaine cohésine, permettant ainsi l'assemblage non-covalent de complexes AcM-Ag. Ainsi, des complexes non-covalents anti-CD40-Ag ou anti-Langerine-Ag du virus influenza ont induit l'expansion in vitro de lymphocytes T CD4+ et CD8+ spécifiques, et la production d'anticorps chez la souris. De même, le ciblage de ces Ag via DCIR, récepteur exprimé sur différentes populations de CD humaines, ont induit l'expansion de lymphocytes T CD8+ mémoires in vitro. Enfin, j'ai montré que l'addition de peptides glycosylés flexibles facilite la production d'AcM anti-CD40 fusionnés à 5 peptides du VIH. Ces peptides conservés et immunogènes sont dérivés des protéines Gag, Nef et Pol. Ce prototype vaccinal, testé in vitro, sur des cellules de patients séropositifs, induit l'expansion d'un vaste répertoire de lymphocytes T CD4+ et CD8+ spécifiques et multifonctionnels. Ces cellules T CD8+ cytotoxiques sont capables de supprimer la réplication du VIH in vitro. En conclusion, ce travail a permis de développer plusieurs prototypes de vaccins ciblant les CD et a démontré leur potentiel à induire des réponses immunes efficaces, justifiant leur application à visée préventive et thérapeutique. / Dendritic cells (DCs), as the most potent antigen-presenting cells, have a pivotal role in the initiation, regulation and maintenance of immune responses against cancers and pathogens. Targeting antigens (Ag) directly to DCs via anti-DC receptor monoclonal antibody-antigen (mAb-Ag) fusion proteins is a promising approach to vaccine development and has been shown to induce potent immunity in animal models. Thus, I developed mAbs fused to a dockerin domain and antigens fused to a cohesin domain, enabling non-covalent assembly of mAb-Ag complexes particularly when direct mAb-Ag fusions could not be produced. Delivery of influenza Ags to CD40 and Langerin via these non-covalent complexes, respectively expanded Ag-specific CD4+ and CD8+ T cells in vitro and elicited Ag-specific antibody responses in mice. Similarly, targeting influenza Ags in vitro with such complexes to DCIR on various human DC subsets expanded Ag-specific memory CD8+ T cells. Finally, I found that flexible glycosylated peptide linkers enabled production of an anti-CD40 mAb fused to a string of 5 conserved T cell epitope- rich regions of HIV Gag, Nef and Pol. In vitro, this prototype vaccine expanded a broad repertoire of Ag- specific and multifunctional CD4+ and CD8+ T cells from HIV-infected patients. These cytotoxic expanded CD8+ T cells were effective in suppressing in vitro HIV replication. In conclusion, our work facilitated the development of prototype DC-targeting vaccines and demonstrated their potential to induce effective immune responses, supporting their use for preventive and therapeutic applications.

L'allergie respiratoire au pollen de bouleau affecte un nombre important de personnes dans le monde. Il est estimé que 100 millions d'individus sont sensibilisés à l'allergène majeur du pollen de bouleau nommé Bet v 1. L’immunothérapie allergénique, basée sur l'administration répétée de l'allergène incriminé, permet la rééducation du système immunitaire du patient d’un profil TH2 vers un profil TH1/Treg et à terme la diminution des symptômes allergiques. Ces travaux de thèse avaient donc pour finalité de produire et de caractériser l'allergène recombinant Bet v 1, à des fins d’immunothérapie allergénique. Dans ce cadre, différentes méthodes analytiques ont été développées et appliquées afin d'optimiser le procédé de production via l'élimination de différentes impuretés liées au produit ou au procédé de production et de documenter la structure de l’allergène. En particulier, l'utilisation de la spectrométrie de masse a permis la détermination de la masse exacte de l'allergène ainsi que la vérification complète de sa séquence en acides aminés. Les travaux en spectrométrie de masse ont également contribué aux détections et identifications de diverses impuretés et produits de dégradations et ont ainsi conduit à plusieurs optimisations du procédé industriel de production de l'allergène recombinant. Les activités immunologiques de certains produits de dégradations ont également été investiguées. La structure tertiaire (spatiale) de l'allergène a été déterminée par diffraction aux rayons X. Enfin, ces travaux ont permis de documenter la qualité de l'allergène recombinant rBet v 1 afin i) de l'établir comme substance de référence pour la Pharmacopée Européenne et ii) de procéder à une étude clinique d’immunothérapie allergénique de phase II auprès de 483 patients allergiques au pollen de bouleau. / Respiratory allergy to birch pollen affects a large number of people in the world. It is estimated that 100 million people are sensitized to the major allergen from birch pollen, namely Bet v 1. Allergen immunotherapy, based on the repeated administration of the allergen of interest, allows the modification of the patient's immune response from a TH2 to a TH1/Treg pattern and thus the reduction of allergic symptoms. This study was therefore aimed to produce and characterize the recombinant Bet v 1 (rBet v 1) allergen, for immunotherapy purpose.In this context, various analytical methods have been developed and applied in order to optimize the production of rBet v 1 via the reduction of process or product-related impurities as well as to document the quality of the purified allergen. In particular, the use of mass spectrometry has allowed the determination of the exact mass of the intact allergen and the complete verification of its amino acid sequence. Mass spectrometry data have also contributed to the detection and identification of impurities and degradation products and have therefore led to several optimizations of the industrial process for the production of the recombinant allergen. Immunological activities of certain degradation products were also investigated and the allergen tertiary structure was determined by X-ray diffraction. Finally, this study was decisive in order i) to establish rBet v 1 as a chemical reference substance for the European Pharmacopoeia as well as ii) to perform a phase II clinical study conducted in 483 patients with birch pollen-induced rhinoconjunctivitis.