PhD Faculty of Health Sciences, University of the Witwatersrand, 2009. / HIV/AIDS was discovered almost a quarter of a century ago and has so far claimed the lives of more than 25 million people worldwide. Developing countries remain disproportionately affected, with sub-Saharan Africa contributing more than two-thirds of infections globally. Sexual transmission is the primary route of HIV/AIDS acquisition, and women bear the greatest burden of this pandemic. We are now at a stage where biotechnological advances are needed that can either cure HIV/AIDS, stimulate the immune system to produce anti-HIV-antibodies by vaccination, or prevent HIV infections. One of these advances has been the development of various microbicides. However, a lack of effective drug delivery systems for these agents has remained as a rate-limiting step towards successful HIV prevention. In an attempt to overcome this problem, this study aimed at designing and developing a novel intravaginal bioadhesive polymeric device (IBPD) as a delivery system to effectively deliver a microbicide {polystyrene sulfonate (PSS)} and antiretroviral (ARV) {3'-azido-3'-deoxythymidine (AZT)} combination to the vagina. The development of a successful intravaginal microbicidal drug delivery system requires the design of a formulation to deliver the microbicide-ARV combination in a safe, effective, and consistent manner. The first step therefore was to undertake extensive preliminary screening studies on various polymeric materials using a one variable at a time (OVAT) approach to find suitable polymers for developing an IBPD. Initially 18 biodegradable and biocompatible polymers were employed to produce 62 formulations that were further screened through the OVAT approach to result in 15 lead formulations. Two major concerns of this study were the attainment of satisfactory residence time of the IBPD in the vagina as well as the ability of the IBPD to contain and release the microbicide-ARV in a controlled manner. Therefore, optimization of the IBPD was based on these two requirements for which proper matrix integrity was a pre-requisite. Artificial neural networks (ANNs), a computational technique that is able to simulate the neurological processing ability of the human brain through mathematical modeling, was employed for optimization. The ANN approach confirmed that 5 of the 18 studied polymers could be suitable for the development of an optimized IBPD. To finally attain good vaginal retention for the developed delivery system, extensive bioadhesivity testing was undertaken on the optimized device. Thorough in vitro and ex vivo bioadhesivity analysis was conducted using physicomechanics and computational structural modeling. Allyl penta erythritolcrosslinked poly acrylic acid (APE-PAA) appeared to contribute most to the bioadhesivity. Apart from being employed as a matrix component, PAA was further used as a coating agent to achieve extended bioadhesivity within the posterior fornix of the vagina. Since prolonged release and suitable permeation of the microbicide-ARV across the vaginal tissue was a critical requirement of this study, the device was designed to provide a controlled and prolonged drug release. Prolonged release for up to 72 days was achieved. Furthermore, the design was constructed to ensure that the released drug could permeate into the vaginal tissue and be retained substantially. This was determined by measuring drug flux through ex-vivo permeation studies using freshly excised pig vaginal tissue in a Franz Diffusion Cell (FDC) apparatus. The ultimate aim of the study was to have the IBPD well accommodated in the vagina for successful prevention of STIs and HIV infection. Achievement of this aim was ensured by undertaking extensive in vivo studies in Large White pig model. The IBPDs were inserted under anaesthesia into the posterior fornix of the vagina, using a novel applicator. To detect the retention of the IBPDs and determine their sequential biodegradation pattern in the vagina, X-ray imaging was employed, using radio-opaque Barium Sulphate (BaSO4). To demonstrate that the developed drug delivery system acted locally and was only minimally absorbed systemically, blood samples were taken from the jugular vein of each pig at pre-determined time intervals and subjected to UPLC analysis. The drug content in the vaginal tissue at the end of the study was also determined. Histopathological evaluation was carried out on vaginal epithelium to access the potential for toxicity of the IBPDs. The drug content analysis revealed that greater amounts of AZT and PSS were retained in the vaginal tissue with relatively small quantities (AZT:17%; PSS:13%) crossing into the systemic circulation. The results from the toxicity studies showed that the IBPDs were safe for use. This suggests that the developed drug delivery system (the IBPD) may be suitable for application in the prevention of STIs and HIV infections.

Lorsque la protéine GILZ (glucocorticoid-induced leucine zipper) est surexprimée dans les cellules dendritiques (DC), elles acquièrent un phénotype tolérogène vecteur d’une fonction immunorégulatrice, notamment par l’induction de lymphocytes T CD4+ régulateurs de type Tr1. Au niveau moléculaire, GILZ peut être induite dans les DC par des signaux extracellulaires anti-inflammatoires comme l’IL-10 et le TGF-b, mais aussi par un cocktail de cytokines pro-inflammatoires (PGE2, IL-6, IL-1b, TNF).L’expression de GILZ dans les DC peut être dérégulée dans des immunopathologies qui reposent sur un déséquilibre du ratio T CD4+ effecteurs / T CD4+ régulateurs comme dans les allergies médiées par les IgE, les cancers.Compte tenu du rôle central joué par les DC dans la mise en œuvre des réponses immunitaires et leur dérégulation dans les infections virales chroniques, nous avons exploré l’expression et la fonction de la protéine GILZ dans les DC dans le cas de l’infection par le VIH-1, chez des patients en phase chronique et sous trithérapies efficaces mais aussi chez des patients elite controllers qui contrôlent naturellement l’infection et préservent des DC immunogènes tout en contrôlant le niveau d’inflammation. Nos données apportent des connaissances nouvelles sur les mécanismes de modulation des DC dans l’infection chronique par le VIH-1, entre l’immunogénicité bénéfique des DC d’elite controllers qui expriment faiblement GILZ et la tolérance délétère des DC de patients sous trithérapies efficaces qui expriment fortement GILZ. Ces découvertes peuvent potentiellement trouver des applications en vaccination, les DC jouant également un rôle crucial dans des stratégies vaccinales. / When the GILZ (glucocorticoid-induced leucine zipper) protein is overexpressed in dendritic cells (DC), they acquire a tolerogenic phenotype that support an immunoregulatory function, notably inducing regulatory CD4+ T cells of a Tr1 type. At the molecular level, GILZ can be induced by anti-inflammatory extracellular signals like IL-10 and TGF-b, as well as by some pro-inflammatory signals (PGE2, IL-6, IL-1b, TNF).A deregulation of GILZ expression in DC has been reported in immune pathologies relying on a disequilibrium between effectors and regulatory CD4+ T cells like allergic diseases and cancers.Considering the central role played by DC in the implementation of immune responses and their deregulation in chronic viral infections, we explored GILZ expression and function in DC in the context of HIV-1 infection, in chronically HIV-1-infected patients under efficacious antiretroviral therapies and in elite controllers that naturally control the infection and preserve immunogenic DC while controlling the level of inflammation. Our data bring new knowledge on the mechanisms of modulation of DC in the chronic infection by HIV-1, between beneficial immunogenicity of DC from elite controllers that barely express GILZ and deleterious tolerance of DC from patients under efficacious antiretroviral therapies that strongly express GILZ. These discoveries may potentially found applications in vaccination, DC also playing a crucial role in vaccine strategies.