Les nouveaux immunosuppresseurs utilisés en transplantation rénale depuis le début des années 2000 :mycophenolate mofetil (MMF), tacrolimus, Neoral (nouvelle formulation de la cyclosporine-Sandimmun) et les inhibiteurs du mTOR ont amélioré le contrôle du risque de rejet, ainsi que la survie du greffon. Ces progrès sont contrebalancés par un risque accru d’infections. Nous avons évalué l'impact des nouveaux immunosuppresseurs sur les défenses humorales anti-infectieuses en 2 parties. Pour la première partie, nous avons étudié les réponses humorales primaires contre des antigènes protéiques. La 1ère étude montre que les réponses après l’administration d’un anticorps monoclonal anti-CD3 (OKT3) en induction sont profondément et additionnellement inhibées par le MMF comparé à l'AZA, et par le Neoral comparé au Sandimmun, ce qui empêche la neutralisation de ce puissant immunosuppresseur. La 2ème étude analyse les réponses au vaccin adjuvanté contre l’influenza H1N1 pandémique de 2009. Seuls 44% des transplantés rénaux ont développé une séroconversion, contre 57% de patients hémodialysés et 90% de contrôles sains. Nous observons aussi que le MMF limite l’augmentation des titres d’anticorps après vaccination par rapport à l’AZA. <p>Pour la seconde partie, nous observons l’évolution de composants de l’immunité humorale et innée au cours de la 1ère année de greffe. La 3ème étude montre que l’incidence de l’hypogammaglobulinémie atteint 45% à 3 mois de greffe, et est encore de 30% à 1 an. Les taux de MBP diminuent progressivement ce qui augmente le risque de sepsis et d’infection virale (CMV), tandis que l’hypogammaglobulinémie combinée :IgG + [IgA ou IgM] est corrélée avec une incidence élevée d’infections précoces (86%, RR :2, P=0.048), surtout d’origine respiratoire. La 4ème étude, indique que les immunosuppresseurs diminuent intensément les titres en Ac spécifiques induits avant la greffe :les Ac anti-pneumococciques, de nature polysaccharidique (Ac T-indépendants) et surtout les Ac anti-Anatoxine tétanique, de nature protéique (Ac T-dépendants). L’ensemble de notre observation conclut que les immunosuppresseurs actuels, et plus particulièrement le Mycophenolate diminuent fortement la production et le maintien des immunoglobulines, et ont un impact infectieux important. Il en découle que les stratégies de vaccination recommandées méritent d'être appliquées et que la vigilance à l'égard des pathogènes doit être implémentée. <p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Das Neuroblastom ist ein vom sympathischen Nervensystem ausgehender neuroektodermaler maligner Tumor des Kleinkindesalters. Bei über 50% der Neuroblastom-Ersterkrankungen liegt bereits das disseminierte Stadium 4 vor, das eine infauste Prognose hat. Eine wirksame Behandlung des Stadium 4 Neuroblastoms stellt deshalb nach wie vor eine der größten Herausforderungen der pädiatrischen Onkologie dar: Die Gesamtüberlebensrate von 20-25% der Kinder, die an dieser bösartigen Krankheit leiden, konnte während der letzten zwei Jahrzehnte trotz neuer Chemotherapie-Protokolle nicht wesentlich verbessert werden. Aus diesem Grund gibt es zunehmend Bestrebungen sich um Therapiealternativen zu bemühen. In dieser Arbeit werden die derzeit möglichen immunologischen Strategien zur Behandlung des Neuroblastoms abgehandelt. / Neuroblastoma is a neuroectodermal malignancy of early childhood derived from sympathetic nervous tissue. At initial diagnosis over 50% of patients present with disseminated stage 4 disease which has a dismal prognosis. Effective treatment of patients with stage 4 neuroblastoma remains a major challenge in pediatric oncology. Despite novel therapeutic approaches including chemotherapy and autologous stem cell transplantation the overall survival rate of only 20-25% did not improve over the last two decades. Therefore, a lot of effort has been made to develop novel alternative therapies. This thesis summarizes possible immunotherapeutic strategies for the treatment of neuroblastoma.

Das Humane Immundefizienzvirus (HIV) ist der Erreger von AIDS und als Zoonose von den Schimpansen (HIV-1) bzw. den Rauchmangaben (HIV-2) auf die menschliche Population übergesprungen. Diese Primatenspezies sind die natürlichen Wirte für die simianen verwand-ten Viren SIVcpz bzw. SIVsm. Die nicht-natürlichen Virus-Wirt-Beziehungen der Immundefizienzviren resultieren in einem pathogenen Verlauf, wie HIV im Menschen und SIVmac in Rhesusmakaken. Die natürlichen Wirte Rauchmangaben, Schimpansen, Afrikanische Grüne Meerkatzen (AGM) und viele mehr entwickeln hingegen kein simianes AIDS. Dies erfolgt trotz lebenslanger Infektion mit SIV und einer zur HIV-Infektion im Menschen äquivalenten Viruslast. Die natürlichen Wirte weisen darüber hinaus keine Immunantwort gegen das virale Kernprotein Gag (gruppen-spezifisches Antigen) auf, was ein früh und zahlreich gebildetes Protein während der Virusreplikation ist. Die fehlende humorale Immunantwort könnte die natürlichen Wirte vor Aktivierung des Immunsystems und dadurch auch vor sAIDS bewahren. Frühere Versuche in AGM mit injiziertem SIVagmGag-Protein zeigten zwar, dass die Induktion einer humoralen Immunantwort gegen SIVagmGag möglich ist, diese aber schon nach kurzer Zeit wieder absinkt. Des Weiteren bildete sich durch Infektion mit SIVagm in den Tieren keine anamnestische Immunantwort heraus, die für die Erkennung von gleichen Epitopen maßgeblich ist. Es scheint ein Unterschied in der Erkennung von exogenem injiziertem SIVagmGag-Protein zu endogenem, durch das Virus selbst gebildetem, Protein in den AGM zu bestehen. Folglich wurde die Hypothese aufgestellt, dass eine Immunisierung mit SIVagmGag-DNA unter Umgehung des Unterschieds in der Proteinprozessierung eine anamnestische Immunantwort in den AGM induziert. Um diese Hypothese zu testen und die Gag-Immunreaktion in Abwesenheit anderer viraler Gene bezüglich der Pathogenität zu evaluieren, wurden codonoptimierte SIVagmGag- und SIVmacGag-DNA Immunisierungsvektoren generiert. Die Proteinexpression wurde in vitro und die Immunogenität in Balb/c und C57Bl/6 Mäusen getestet. Jeweils eine Gruppe von vier AGM erhielt bioballistisch SIVagmGag-DNA bzw. SIVmacGag-DNA. Als Kontrollen dienten mit codonoptimierter DNA immunisierte Rhesusmakaken sowie mit Leervektor immunisierte Primaten beider Spezies. Als Kostimulanz wurde zusätzlich jeweils speziesspezifische gmcsf DNA verwendet. Im Gegensatz zu den Rhesusmakaken konnte in den AGM durch DNA-Immunisierung keine zelluläre und nur eine schwache, transiente humorale anamnestische Immunantwort nach Infektion induziert werden, obwohl beide Gag-Proteine endogen produziert wurden. Daher kann die fehlende anamnestische Immunantwort der AGM nach Immunisierung mit Gag-Protein vermutlich nicht auf Unterschiede in der Proteinprozessierung und -erkennung (endogen versus exogen) zurückgeführt werden. Die Ergebnisse dieser Studie deuten an, dass während der Infektion dieses natürlichen Wirtes eine aktive Unterdrückung der anti-Gag-Antikörperantwort stattfindet, möglicherweise induziert durch eine Anergie in Gag-spezifischen T-Helferzellen. / The causative agents of AIDS, the human immunodeficiency viruses (HIV-1 and HIV-2), were transmitted zoonotically to the human population from the natural hosts of the related simian immunodeficiency viruses SIVcpz (chimpanzees) and SIVsm (sooty mangabeys), respectively. In contrast to the outcome of infections in non-natural hosts (e.g. HIV in humans, SIVmac in rhesus macaques), the natural hosts of SIV do not develop simian AIDS-like symptoms despite life-long infection with virus loads matching those seen in HIV-infected humans. Many such natural host primates infected with SIV, such as SIVagm-infected African green monkeys (AGMs), fail to mount an antibody response to the intact viral core protein Gag (group-specific antigen), a protein produced extensively during infection. It has been postulated that this lack of an immune response to Gag could protect the natural hosts from immunopathological effects and therefore from simian AIDS. Previous studies in AGMs indicated that Gag protein produced endogenously during infection is ''seen'' by the immune system differently than that introduced exogenously by protein immunisation, possibly through differences in processing or through specific tolerance at the T-cell level. To address these possibilities, primate studies were performed in which the responses in the natural and non-natural hosts to endogenously produced Gag protein in the absence of other viral genes were compared and the influence of such endogenous priming on the immune response to infection was evaluated. This was achieved by bioballistic immunisation of AGMs and rhesus macaques with codon-optimised DNA coding for SIVagm or SIVmac Gag protein delivered together with DNA coding for the species-specific GM-CSF cytokine. Prior to the primate studies, the DNA constructs were evaluated in BALB/c and C57Bl/6 mice for immunogenicity and protein expression. In contrast to the rhesus macaques, SIVagmGag DNA immunisation of African green mon-keys generally failed to prime for an anamnestic cellular immune response and primed for only a weak, transient anamnestic humoral response to the protein upon infection, despite the proteins resulting from both immunisation and infection being produced endogenously. Differences in protein processing and recognition (endogenous versus exogenous) do not therefore appear to account for the lack of priming for an anamnestic immune response seen using a protein immunogen. Rather, the results seem to indicate that an active suppression of the anti-Gag immune response may occur during infection of this natural host of SIV, possibly by the induction of anergy in Gag-specific Th cells.

Der Einzeller Eimeria tenella zählt zu den hochpathogenen Parasiten des Haushuhns und ist Verursacher der Blinddarm-Kokzidiose, eine Erkrankung, die zu hohen ökonomischen Belastungen in der Geflügelindustrie führt. Zur Zeit existiert noch kein Impfstoff, der auf Basis von einzelnen Antigenen wirksam ist. Insbesondere die Identifizierung und Charakterisierung von sekretorischen Proteinen, welche an der Parasit-Wirtszell-Interaktion beteiligt sind, könnte einen Beitrag zur Entwicklung einer Immunprophylaxe darstellen. Ziel dieser Arbeit war es, sekretorische E. tenella-Proteine zu identifizieren und ausgewählte cDNA-Sequenzen in DNA-Immunisierungsstudien zu überprüfen. Um ein DNA-Immunisierungsprotokoll für Hühner zu erarbeiten, wurden die cDNAs von drei schon bekannten E. tenella-Antigenen alleine oder in Fusion zur cDNA des stabilisierenden enhanced green fluorescence protein (EGFP) in zwei unterschiedliche DNA-Immunisierungsvektoren (pCDNA3, pVR1012) kloniert. Die Überprüfung der Serokonversion zeigte, dass nach DNA-Immunisierung von Hühnern mit beiden Vektoren bzw. mit und ohne EGFP-Fusion vergleichbare Immunantworten induziert wurden. D.h. mit dem etablierten DNA-Immunisierungsprotokoll konnte eine Expression heterologer (Eimerien) Gene im Huhn erzielt werden. Um neue sekretorische E. tenella-Proteine zu identifizieren wurden bioinformatische und experimentelle Methoden eingesetzt. Über die bioinformatischen Analysen wurden aus E. tenella-expressed sequence tag (EST)-Datenbanken 54 putativ sekretierte E. tenella-Sequenzen extrahiert für die aufgrund ihrer beschriebenen Funktion und Lokalisierung eine Beteiligung an der Wirts-Parasit-Interaktion abgeleitet wurde. Mittels funktioneller Komplementierung in Hefen konnten aus einer E. tenella-Sporozoiten-cDNA-Bank 25 sekretorische Sequenzen identifiziert werden. Die cDNA-Sequenzen von 13 neu identifizierten sekretorischen Proteinen wurden in DNA-Immunisierungsstudien überprüft. Die Ergebnisse zeigten, dass beide Methoden geeignet sind, um sekretorische Proteine von E. tenella zu identifizieren. Aufgrund der Erkenntnisse dieser Arbeit, sollten jedoch weitere verbesserte Immunisierungsprotokolle erarbeitet werden, um die immunprotektive Wirkung der isolierten Kandidaten-Antigene von E. tenella in Hühnern zu überprüfen. Wie die vorliegende Arbeit bestätigt, eignet sich die Methode der DNA-Immunisierung vermutlich als Grundlage solcher Verfahren, wobei zukünftig die Induktion der essentiellen intestinalen Immunantwort im Vordergrund stehen sollte. / The protozoan parasite Eimeria tenella is highly pathogenic in chickens and causes caecal coccidosis that contribute to high economic losses in poultry farming. At the moment no vaccine based on a single antigen is available. The identification and characterization of proteins that are involved in host parasite interaction could particularly contribute to the development of immunoprophylactic control strategies. In order to establish a DNA immunisation protocol, cDNA of three already known E. tenella antigens were subcloned alone or in fusion to the stabilising fusion partner enhanced green flurescence protein (EGFP) into two different DNA immunisation vectors (pCDNA3, pVR1012). Following DNA immunisation of chickens using both vectors with or without EGFP fusion, respectively, the induction of comparable immune responses were shown by serum conversion. This implies it was possible to achieve heterologous (Eimeria) gene expression in chickens with the established immunisation protocol. In order to identify new E. tenella secretory proteins bioinformatic and experimental approaches were used. Following bioinformatic analysis 54 putatively secretory E. tenella sequences were identified for which roles in host parasite interaction were surmised, due to their localisation and function. The identification of secreted proteins from a E. tenella sporozoite cDNA library using a functional complementation assay in yeast resulted in 25 unique sequences. The cDNA of 13 newly identified secretory proteins were tested in DNA immunisation studies. The results showed that both methods are capable of identifying secretory proteins of E. tenella. Owing to the results presented here, new immunisation protocols should be established to test the immune protective capacity of candidate antigens of E. tenella in chickens. The present study confirmed the method of DNA immunisation as a basic tool for such new protocols. In future, however, DNA immunisation protocols should focus on the induction of essential intestinal immune responses.

Chitinasen sind wichtigen Moleküle im Lebenszyklus parasitischer Fadenwürmer und bedeutende Medikamenten- und Impfstoffziele. Hier wurden drei Chitinasesequenzen (I, II und III) durch Charakterisierung von 9 Klonen aus einer Genbank von Acanthocheilonema viteae gefunden. Die Anzahl der drei sehr homologen Chitinasegene wurde durch Southern-Blot bestätigt. Die größten Unterschiede sind in den Exons zu finden, die die Serin-Threonin-reiche Domäne der Chitinasen codieren. Diese Domäne ist in Sequenz III ca. 10fach länger als in Sequenz I. Sequenz I und III haben Eigenschaften eines transkribierten Genes: ein Startcodon, ein offener Leserahmen, ein Stopcodon und ein Polyadenylierungssignal. Sequenz II fehlt das erste Exon mit dem Startcodon. Bei Durchmusterung einer cDNA-Bibliothek adulter A. viteae Würmer bzw. L3 Stadien, sowie durch Reverser Transkriptase-PCR (RT-PCR) wurden in den Mikrofilarien, L3 und L4 Transkripte für Gen I gefunden, jedoch nicht für Gen III. Das N-terminale Fragment der A. viteae-Chitinase wurde in ein Expressionsplasmid ligiert und in E. coli exprimiert. Ungefähr 80 % der exprimierten Chitinase lagen in Einschluss-Körpern vor. Dieser unlösliche Anteil konnte unter denaturierenden, der lösliche Anteil unter nativen Bedingungen aufgereinigt werden. Die aus den Einschluss-Körpern aufgereinigte Chitinase zeigte im Vergleich zur löslichen Chitinase eine 13fach verminderte Aktivität. Vom aktiven Zentrum der Chitinase wurden Peptide synthetisiert und parallel mit Chitinase aus den Einschluss-Körpern für Vakzinierungsexperimente im A. viteae / Meriones unguiculatus-Filarien-Modell genutzt. Die Reduzierung der Wurmlast um 29 % nach einer Immunisierung mit rekombinantem Protein zeigte eine tendenziell schützende Kapazität des Proteins. In zwei unabhängigen Experimenten konnte nach Immunisierung mit zwei synthetischen Peptiden (P1 und P2) eine bedeutende Reduktion der Wurmlast beobachtet werden. In der P1-Gruppe gab es eine gleichmäßige Reduktion der Mf-Last, die nur im zweiten Experiment signifikant war (39 %, p > 0,05 und 45 %, p < 0,05). In der P2-Gruppe konnte in einem von zwei Experimenten eine signifikante Reduktion der Mf-Last erzielt werden (75 %, p < 0,05). Diese Ergebnisse lassen vermuten, daß Filarien-Chitinasen potenzielle Ziele für Impfstoffe sind. / Chitinases are enzymes that break down chitin to its monomers. They are important molecules in the life cycle of parasitic nematodes representing important drug and vaccine targets. Here, three chitinase sequences (I, II and III) were obtained by characterising nine clones from a genomic library of Acanthocheilonema viteae. Southern blots confirmed the existence of three chitinase genes in A. viteae. The organisation of all three genomic sequences is similar, with the greatest difference occurring in exons encoding the serine-threonine rich domain of chitinases: this domain is about ten times larger in sequence III compared to I, but is absent in sequence II. Sequence I and III had features of regularly transcribed genes: start ATG, open reading frame, stop codon and polyadenylation signal. Sequence II lacked the first exon with start ATG. Screening of cDNA libraries from adult female A. viteae worms and L3 (third-stage larvae), as well as reverse transcriptase PCR (RT-PCR) ! showed transcripts in uterine microfilariae, L3 and L4 (fourth-stage larvae) for gene I only. The N-terminal fragment of A. viteae chitinase was cloned into an expression vector and expressed in Escherichia coli. About 80% of the expressed chitinase were found in inclusion bodies and were purified under denaturing conditions. The soluble fraction was about 20% and could be purified under native conditions. Chitinase purified from inclusion bodies was 13-fold less active compared to soluble chitinase. Synthetic peptides (P1 and P2) were designed from the active site of A. viteae chitinase, and used in parallel with chitinase from inclusion bodies in vaccination experiments using the A. viteae / Meriones unguiculatus filariasis model. Vaccination with recombinant protein led to a 29 % significant reduction in adult worm burden in a single experiment. Vaccination with P1 and P2 led to an overall non significant reduction in adult worm burden in two independent experiments. In the P1 group, there was a consistent reduction (39%, p>0.05 and 45%, p

This study investigated caregivers' perceptions with regard to vaccine-preventable diseases in terms of six constructs of the Health Belief Model. A qualitative research design that is explorative, descriptive and contextual in nature was employed in order to understand and describe the perceptions influencing access and utilisation of services that lead to missed immunisation opportunities and consequently outbreaks of vaccine-preventable diseases. The data-collection techniques were individual unstructured in-depth interviews, field notes and clinical records. The sample consisted of twenty two (N=22) caregivers who volunteered to be interviewed. The study has highlighted that caregivers' perceptions or cognitive factors play an important role for having children in completing immunisation schedule to protect the public from vaccine-preventable diseases. It is recommended that mass media programmes are needed to address the role of vaccines in reducing high morbidity and mortality rates caused by vaccine preventable diseases and improvement in access to immunisation services. / Health Studies / MPH (Health Studies)

This study investigated caregivers' perceptions with regard to vaccine-preventable diseases in terms of six constructs of the Health Belief Model. A qualitative research design that is explorative, descriptive and contextual in nature was employed in order to understand and describe the perceptions influencing access and utilisation of services that lead to missed immunisation opportunities and consequently outbreaks of vaccine-preventable diseases. The data-collection techniques were individual unstructured in-depth interviews, field notes and clinical records. The sample consisted of twenty two (N=22) caregivers who volunteered to be interviewed. The study has highlighted that caregivers' perceptions or cognitive factors play an important role for having children in completing immunisation schedule to protect the public from vaccine-preventable diseases. It is recommended that mass media programmes are needed to address the role of vaccines in reducing high morbidity and mortality rates caused by vaccine preventable diseases and improvement in access to immunisation services. / Health Studies / MPH (Health Studies)

De nombreuses études suggèrent que linhibition de la voie alterne du complément de lhôte est nécessaire aux parasites hématophages pour leur permettre daccomplir leur repas sanguin. Une revue décrivant ces études a été publiée dans Developmental and Comparative Immunology (Schroeder et al., (2009) Dev. Comp. Immunol. 33, 5-13). Plusieurs études suggèrent que linhibition de la voie alterne de lhôte par les protéines salivaires de tiques est importante pour lacquisition du repas sanguin et la transmission subséquente de pathogènes à lhôte infesté. Confortant cette hypothèse, une protéine salivaire capable dinhiber la voie alterne du complément a été clonée chez la tique américaine Ixodes scapularis (Valenzuela et al., (2000) J. Biol. Chem. 275, 18717-18723). Cette protéine, appelée Isac, ne présente aucune homologie avec les autres molécules anti-complément connues à ce jour, suggérant que cette protéine a été acquise au cours de lévolution par un mécanisme dévolution convergente. En plus de cet aspect fondamental, Isac représente un candidat antigénique prometteur pour le développement dun vaccin anti-tique potentiellement capable dinduire le rejet de la tique et/ou de prévenir la transmission des pathogènes. Le but initial de cette thèse était didentifier le ou les homologue(s) de la protéine Isac de la tique américaine Ixodes scapularis chez la tique européenne Ixodes ricinus. De façon intéressante, deux séquences différentes ont été isolées du transcriptome des glandes salivaires de la tique I. ricinus (Daix et al., (2007) Insect Mol. Biol. 16 (2), 155-166). Lexpression de ces séquences a révélé quelles codent pour des protéines secrétées capables dinhiber la voie alterne du complément. Ces protéines ont été appelées IRAC I et II pour « Ixodes ricinus anti-complement protein I and II ». La caractérisation des IRACs à laide danticorps monoclonaux a permis de révéler que ces deux protéines sont exprimées de façon constitutive au sein des glandes salivaires de la tique Ixodes ricinus et sont sur-exprimées au cours du repas sanguin. Létude de lexpression des protéines IRAC I et IRAC II au sein de la population dIxodes ricinus a révélé que ces deux protéines sont des paralogues codés par des gènes différents et non par des allèles dun même locus. Enfin, des analyses phylogéniques portant sur les différentes séquences codant pour les protéines homologues à Isac isolées chez les tiques Ixodes scapularis, Ixodes ricinus et Ixodes pacificus ont révélé que les tiques appartenant au complexe Ixodes ricinus codent pour une famille encore non décrite de molécules anti complément qui se sont diversifiées au cours de lévolution par un processus de sélection darwinienne positive. Les analyses phylogéniques des IRACs suggèrent que ces séquences ont subi une diversification par un processus de pression de sélection darwinienne positive, menant probablement à des molécules possédant des propriétés biologiques différentes. Dans la seconde étude, nous avons testé lhypothèse de travail selon laquelle chaque paralogue pourrait posséder des activités inhibitrices différentes à lencontre du complément de différentes espèces dhôtes naturels, contribuant ainsi à élargir le spectre dhôte des tiques I. ricinus. Les résultats obtenus démontrent que cette hypothèse est correcte (Schroeder et al., (2007) Microbes Infect. 9 (2), 247-250). Dans la troisième et dernière étude de ce manuscrit, nous avons testé le potentiel des protéines IRACs comme candidat antigénique pour le développement dun vaccin anti tique. Des recombinants de lherpèsvirus bovin 4 (BoHV-4) exprimant IRAC I ou IRAC II ont été produits. De façon intéressante, nous avons observé que bien que les recombinants BoHV-4 expriment de hauts taux de protéines IRACs fonctionnelles in vitro, les lapins immunisés à laide des recombinants BoHV-4 exprimant les IRACs ne développent pas de réponse humorale détectable à lencontre des transgènes. Dans le but daugmenter limmunogénicité des IRACs exprimés comme transgène, une seconde génération de recombinants BoHV-4 a été produite. Ceux-ci induisent lexpression des IRACs sous la forme de protéines de fusion transmembranaires à la surface des cellules infectées. Linoculation de ces recombinants à des lapins a engendré le développement dune forte réponse humorale à lencontre des IRACs. Néanmoins, cette réponse immune na pas engendré deffet majeur sur le repas sanguin de femelles Ixodes ricinus placées sur les lapins immunisés. / An increasing number of studies demonstrate that inhibition of host complement activation is crucial for completion of the blood feeding process of hematophagous parasites. A review of these studies has been published in Developmental and Comparative Immunology (Schroeder et al., (2009) Dev. Comp. Immunol. 33, 5-13). Several observations suggest that inhibition of the host complement alternative pathway by tick salivary proteins is crucial for the achievement of blood feeding and efficient transmission of the pathogens transmitted by the parasite. Strongly supporting this conclusion, a salivary protein able to inhibit the alternative pathway was cloned from the American tick Ixodes scapularis (Valenzuela et al., (2000) J. Biol. Chem. 275, 18717-18723). Interestingly, this molecule, termed Isac, has no similarity to any previously reported anti-complement molecules suggesting that it has been acquired through a mechanism of convergent evolution. In addition to this fundamental aspect, Isac is also a promising candidate antigen for the development of an anti-tick vaccine potentially able to induce the reject of the tick and/or to prevent the transmission of the pathogens. The initial goal of the present work was to clone the orthologue of Isac from the European tick Ixodes ricinus. Interestingly, two different sequences were isolated from the transcriptome of I. ricinus salivary glands (Daix et al., (2007) Insect Mol. Biol. 16 (2), 155-166). Expression of these sequences revealed that they both encode secreted proteins able to inhibit the complement alternative pathway. These proteins were called I. ricinus anticomplement (IRAC) protein I and II. Further characterization of IRACs using monoclonal antibodies revealed that both proteins are expressed constitutively in I. ricinus salivary glands and are up-regulated during blood feeding. Analysis of a series of individual ticks revealed that all ticks tested express both IRAC I and IRAC II, demonstrating that they are the products of different genes and not of alleles of the same locus. Finally, phylogenetic analyses of the I. ricinus IRAC I and II sequences together with homologues from I. scapularis and I. pacificus demonstrates that ticks belonging to the Ixodes ricinus complex encode a new family of relatively small anti-complement molecules undergoing diversification by positive Darwinian selection. Phylogenetic analyses of IRACs suggested that these sequences were diversifying by a process of positive Darwinian selection, possibly leading to molecules with different biological properties. In the second study, we tested the hypothesis that each paralogue may have different inhibitory activities against the complement of different natural host species, thereby contributing to broaden the host range of I. ricinus ticks. The data obtained demonstrated that this working hypothesis is correct (Schroeder et al., (2007) Microbes Infect. 9 (2), 247-250). In the third and last chapter of the present manuscript, we addressed the potential of IRAC I and II as candidate antigens for the development of an anti-tick vaccine. Bovine herpesvirus 4 (BoHV-4) recombinants expressing IRAC I or II were produced. Interestingly, we observed that although both recombinants expressed high levels of functional IRAC proteins in vitro, our attempts to immunize rabbits against IRACs via infection with these viruses invariably failed. In order to improve the immunogenicity of IRACs expressed as transgene, a second generation of BoHV-4 recombinants was produced. The latter expressed IRACs as transmembrane fusion proteins on cell surface. Comparison of the vaccine potential of BoHV-4 recombinant viruses expressing either secreted or transmembrane IRAC proteins revealed that while the former did not induce a detectable immune response against IRACs, the latter led to high titers of anti-IRAC antibodies. However, the immune response induced against IRACs did not lead to the reject of the tick but only slightly increased the duration of the blood feeding process.

Newborn hearing screening has become an increasingly important element of neonatal care in developed countries whilst only a few fragmented screening programmes are evident in developing countries. The numerous socio-economic, cultural and healthcare barriers in developing contexts do not, however, negate or diminish the need to ensure optimal outcomes for infants with hearing loss through early identification and intervention programmes. South Africa has taken a first step toward addressing this need by publishing a Year 2002 Hearing Screening Position Statement that was produced by the Professional Board for Speech, Language and Hearing Professions of the Health Professions Council of South Africa. Interim recommendations are made toward universal newborn hearing screening programmes in three contexts: well-baby nurseries,; neonatal intensive care units (NICU) and Maternal and Child Health (MCH) clinics through their 6-week immunisation programmes. Although these clinics constitute an unfamiliar hearing screening context, they are essential platforms toward widespread screening of the majority of infants in South Africa. An urgent need therefore exists to ascertain the feasibility of hearing screening programmes at MCH 6-week immunisation clinics in order to guide the future implementation of widespread hearing screening services in South Africa. To attend to this need, an exploratory descriptive design that jointly implements quantitative and qualitative methods in a dominant-less-dominant model of triangulation was utilised to critically describe a screening programme conducted at two MCH clinics in Hammanskraal (a developing, peri-urban South African community). The quantitative methods included a structured interview to compile biographical and risk information; high frequency immittance measurements; hearing screening with OAE and AABR according to specified protocols, and diagnostic assessment of referred infants. The qualitative methods included field notes and critical reflections describing clinics as screening contexts and elucidating interactional processes involved in sustaining programmes. A total number of 510 infant-caregiver pairs were enrolled as subjects during the five-month research period. Results indicate that clinics not only provide a suitable context, but also the possibility of effective collaborations toward facilitating effective initial infant hearing screening programmes. The caregivers and infants who attended the clinics demonstrated significant degrees of socio-economic deprivation. They also reported an increased incidence of risk indicators exacerbating the population’s risk for congenital hearing loss, poor participation in the hearing screening/follow-up process, and subsequent poor involvement in a family-centred early intervention process for infants identified with hearing loss. The screening protocol effectively classified infants into risk categories for hearing loss and established useful norms for high frequency immittance in infants. The efficiency of the programme was acceptable considering the short period of implementation, but inefficient coverage with the AABR and poor follow-up return rates were obtained at the clinics. Despite prevailing barriers, the MCH 6-week immunisation clinics showed promise as platforms for widespread hearing screening programmes for infants in South Africa. The clinical implications and recommendations that emerged from the research conducted in this study were compiled and presented in the form of a preliminary service delivery model for infant hearing screening at MCH clinics. / Thesis (DPhil (Communication Pathology))--University of Pretoria, 2004. / Speech-Language Pathology and Audiology / unrestricted

La vaccination qui est le sujet sur lequel porte cette recherche est une des questions de santé publique les plus importantes; elle fait néanmoins l’objet de nombreuses controverses. Dans le contexte de cette thèse, c’est plutôt l’accès à la vaccination qui est mis en question. La présente recherche vise à analyser une stratégie d’amélioration de la couverture vaccinale à l’aide d’une évaluation de processus extensive en trois étapes faisant suite à une documentation approfondie du contexte. En effet, la recherche analyse les perceptions et les facteurs d’influence de la couverture vaccinale avant l’intervention, les assises conceptuelles et théoriques de cette intervention, l’implantation et la réception de l’intervention et enfin les résultats et les mécanismes mis en œuvre pour les atteindre. Les résultats indiquent que la vaccination s’insère dans l’ensemble des stratégies locales de protection fondées sur des notions endogènes du risque. Ces éléments culturels associés à des facteurs socioéconomiques et aux rapports entre parents et services de santé concourent à expliquer un niveau relativement bas de couverture vaccinale complète de 50% avant l’intervention. L’analyse exploratoire de l’intervention indique que celle-ci intègre une théorie initiale implicite et une philosophie. L’intervention finale était évaluable; cependant, la validation de sa théorie a été compromise par des écarts dans l’implantation. L’approche descriptive montre des taux de réalisation d’activités assez élevés, une atteinte de plus de 95% des cibles et un niveau de réception acceptable, ce qui indique que l’intervention est une stratégie réalisable mais à améliorer. La couverture vaccinale après l’intervention est de 87%; elle est influencée positivement par les niveaux de connaissance élevés des parents et le fait pour les enfants d’être nés dans un centre de santé, et négativement par l’éloignement par rapport au site de vaccination. L’atteinte des résultats suit la procédure principale d’amélioration du niveau de connaissance des parents. Celle-ci est basée sur un mécanisme latent qui est la perception des « opportunités » que fournit la vaccination pour prévenir divers risques sanitaires, sociaux et économiques. Cependant, des approches complémentaires tentent de maximiser les effets de l’intervention en utilisant les pouvoirs conférés aux relais communautaires féminins et la coercition sociale. Cette recherche contribue à éclairer la relation entre l’évaluation du processus et l’analyse de l’évaluabilité, à conceptualiser et opérationnaliser autrement les notions de doses d’intervention administrées et de doses reçues. Sur le plan de la pratique, la recherche contribue à proposer l’amélioration des profils de personnel pour les activités de vaccination et la vulgarisation de la stratégie. Des propositions sont faites pour l’amélioration de l’intervention et l’information des institutions de financement des interventions. / Immunization is one of the most important subjects in public health despite constant on-going controversies. In the context of this research it is more its accessibility that is at issue. This particular research is situated in the perspective of the comprehensive approaches to evaluation and analyses an immunization improvement strategy using a three steps process evaluation after documenting its context. The perceptions and factors influencing immunization coverage before the intervention are assessed, as well as the conceptual and theoretical foundations of the intervention, the implementation and reception of the intervention and mechanisms used to attain the results. The results indicate that immunization is integrated into the local strategies used for protection and grounded in the local concept of risk. Results show also that the context associated with socioeconomic factors and the relations between parents and health services contribute to explain the relatively low complete immunization coverage rate (50.3%) before the intervention. The exploratory analysis indicates that the intervention had an implicit theory and a philosophy. Although the intervention was adequate for an evaluation, discrepancies in the implantation compromised the possibilities of validating its initial theory and philosophy. The descriptive analysis showed that more than 95% of the recipients were reached by the intervention and the received dose of intervention was acceptable indicating that the intervention is a workable strategy that needs to be improved. The complete immunization coverage after the intervention was 87.3%; it was influenced positively by factors including parents’ level of knowledge and the fact that the child was born in a health center, and negatively by the long distances from household to immunization place. The strategies involved in the attainment of the results used parents’ knowledge improvement as a principal procedure. This contains an underlying mechanism mainly related to the perception of the opportunities that immunization permits for preventing various health, social and economic risks. However, complementary approaches tend to maximize intervention outcome by using the power given to female community immunization facilitators and social coercion. The research contributes to highlight the relationship between evaluability assessment and process evaluation, to propose a new conceptual and operational understanding of dose of intervention administered and dose of intervention received. On practical grounds, this research recommended the improvement of the immunization activities staff profile and the widespread adoption of the strategy after it improvement. Indications are provided to improve the intervention and to inform founding agencies.