Development of an improved TB vaccine is hindered by the lack of a correlate of ,~.-r protection. Efficacy of TB vaccines in humans can only be a"S~essed by expensive and time consuming trials within TB endemic areas, which are limited; therefore, it is critical that vaccines with the greatest potential to protect are selected for these trials. Mycobacterial growth inhibition assays (MGIAs) have been developed with the hope that these in vitro functional assays will correlate with protection, which could aid in the selection of the best vaccine candidates. Work in this thesis describes the development and evaluation of different MGIAs for their ability to detect TB vaccine induced mycobacterial growth inhibition. The mycobacterial growth indicator tube (MGIT) MGIA reproducibly demonstrated mycobacterial growth inhibition in splenocytes from BCG vaccinated compared with naive mice, which corresponded with in vivo protection from M. tb challenge. This assay also discriminated between PBMC from naive and BCG/BCG-MVA85A vaccinated macaques. Microarray data showed extensive differential gene expression in splenocyte responses to ex-vivo BCG stimulation between naive and BCG vaccinated mice. T H 1 responses including IFN-y with NOS2 expression were enhanced in BCG vaccinated mice, indicating a possible mechanism for mycobacterial growth inhibition in BCG vaccinated mice. Further investigation into whether the MGIT assay can be used as a correlate of protection from M. tb in humans and animals is warranted.

Francisella tularensis is one of a small group of bacteria recognized for their virulence and potential for use as biological weapons. In this study we utilize a novel approach to identify an immunologically prominent component of F. tularensis that appears to be a promising vaccine candidate. Francisella is an intracellular pathogen that infects cells of the reticuloendothelial system. Other bacteria, such as Brucella spp. have this part of their life cylce in common. However, while mice injected with Brucella spp. survive and produce antibodies to the bacteria which are immunologically reactive not only with Brucella spp. but, also with Francisella. When we vaccinated mice with a B. abortis O-linked polysaccharide (OPS) and then challenged them with 10 LD50F.tularensis LVS, 60% survived. Sera from Brucella OPS-primed/F.tularensis-challenged mice was used to identify immune reactive proteins from F. tularensis. A novel 52 kDa fraction was identified. While vaccination of mice with this partially purified fraction only provided 20% protection to F.tularensis challenged mice, both whole cell extracts and a partially purified soluble fraction (>30kDa) given to Brucella-vaccinated mice were 100% protective. The 52 kDa enriched fraction elicited a rudimentary cytokine burst of nitric oxide in a cell culture of J774.1 macrophages. The 52 kDa fraction was degraded by proteinase K and appeared to decrease in size to 36 kDa in the presence of DNAase, suggesting a possible protein and nucleic acid composition. The host response to F. tularensiss infection is complex, but given the ability of the 52 kDa component to protect against live vaccine challenge, and its apparent ability to elicit a cytokine burst, this component may have potential use in future vaccine production. / xii, 97 leaves ; 29 cm.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

M. Tech. Veterinary Technology. / Aims of this study were to: determine the suitable buffers and optimal concentrations of these buffers, to be used in the ISA 206 B oil-based vaccine formulations that will ensure pH levels of &#x2265%x; 7.0; consistent emulsion type and particle sizes following a storage period of at least 24 months at 4 C ; determine the effects of temperature on the stability of the vaccine formulation during storage ; determine the optimal buffer antigen ratio in the water phase of the ISA 206 B oil-adjuvanted FMD vaccine containing SAT serotypes ; determine the effects of saponin (Q-Vac trade mark) on the buffering capacity during storage, in the ISA 206 B oil-adjuvanted FMD vaccine containing the SAT serotypes; and determine the shelf life of this improved (stabilised) oil vaccine. Previous research by the ARC scientists has shown that the immunity elicited by the ISA 206 oil adjuvanted vaccines could persist up to 50 weeks post vaccination in cattle (Cloete et al., 2008; Hunter, 1996). However, they did not show if this immunity was protective or not. Although it is known within the FMD field that sometimes immunity levels do not always translate into protection against an infection, if protection can be shown - even after vaccination using a stored vaccines - achievement of the above mentioned objectives could enable a once-a-year vaccination regimen in the control zone of RSA. Moreover, this once-a-year vaccination regimen could also substantially reduce the logistical costs involved during vaccination campaigns, compared to the current biannual vaccination regimen. Once the shelf life of the vaccine has been established, the vaccine could also be registered as a stock remedy under the Fertilisers, Farm Feed, Agricultural Remedies and Stock Remedies Act, 36 of 1947, administered by DAFF. The registration of this vaccine could in turn enable the RSA to supply the vaccine to neighbouring South African Development Community (SADC) countries and the rest of African countries where the SAT serotypes occur.

M. Tech. Veterinary Technology. / Aims of this study were to: determine the suitable buffers and optimal concentrations of these buffers, to be used in the ISA 206 B oil-based vaccine formulations that will ensure pH levels of &#x2265%x; 7.0; consistent emulsion type and particle sizes following a storage period of at least 24 months at 4 C ; determine the effects of temperature on the stability of the vaccine formulation during storage ; determine the optimal buffer antigen ratio in the water phase of the ISA 206 B oil-adjuvanted FMD vaccine containing SAT serotypes ; determine the effects of saponin (Q-Vac trade mark) on the buffering capacity during storage, in the ISA 206 B oil-adjuvanted FMD vaccine containing the SAT serotypes; and determine the shelf life of this improved (stabilised) oil vaccine. Previous research by the ARC scientists has shown that the immunity elicited by the ISA 206 oil adjuvanted vaccines could persist up to 50 weeks post vaccination in cattle (Cloete et al., 2008; Hunter, 1996). However, they did not show if this immunity was protective or not. Although it is known within the FMD field that sometimes immunity levels do not always translate into protection against an infection, if protection can be shown - even after vaccination using a stored vaccines - achievement of the above mentioned objectives could enable a once-a-year vaccination regimen in the control zone of RSA. Moreover, this once-a-year vaccination regimen could also substantially reduce the logistical costs involved during vaccination campaigns, compared to the current biannual vaccination regimen. Once the shelf life of the vaccine has been established, the vaccine could also be registered as a stock remedy under the Fertilisers, Farm Feed, Agricultural Remedies and Stock Remedies Act, 36 of 1947, administered by DAFF. The registration of this vaccine could in turn enable the RSA to supply the vaccine to neighbouring South African Development Community (SADC) countries and the rest of African countries where the SAT serotypes occur.

Une compréhension fine et détaillée ciblant les mécanismes d’action de nouvelles molécules adjuvantes sur notre système immunitaire vise de manière directe à l’élaboration de nouveaux vaccins plus ciblés et plus efficaces, mais aussi à élargir nos connaissances quant à l’induction d’une réponse immune protectrice.<p>Au cours de cette thèse nous avons voulu comprendre les modes d’action de deux molécules lipidiques distinctes.<p>La première est le lipide cationique diC14-amidine dont il avait été démontré une action sur les cellules dendritiques en culture par une voie qui restait à élucider. Ce lipide cationique s'organise sous forme de liposomes en milieu aqueux et peut s'associer à de nombreux antigènes. La seconde est un analogue synthétique de l'adjuvant monophosphoryl lipide A (MPL), un dérivé du LPS, nommé CRX-527. À l'instar de sa molécule parente, le CRX-527 active le récepteur TLR4 et est considéré comme un adjuvant potentiel de vaccin ou comme immunostimulant isolé.<p>Au cours de notre travail, nous avons démontré que la diC14-amidine active les cellules cibles via le récepteur TLR4. En effet, l'absence de ce récepteur abolit les réponses induites par le lipide cationique diC14-amidine et la transfection du gène codant pour TLR4 rend répondeuses des cellules qui n'exprimaient pas ce récepteur. De plus, la diC14-amidine active et mature des cellules dendritiques, aussi bien de provenance murine qu'humaine, suggérant qu'elle puisse être utilisée en tant qu’adjuvant. Il avait d’ailleurs été précédemment décrit que l'injection d'un complexe diC14-amidine / allergène chez la souris induisait une réponse immune suffisante pour conférer une protection contre cet allergène. Dans ce contexte, nous avons caractérisé au niveau cellulaire la réponse induite suite à l'injection du complexe diC14-amidine / ovalbumine chez la souris. Cette réponse se manifeste par une production d'IFNγ lors d'une re-stimulation ex vivo par l'antigène OVA. <p>En ce qui concerne la molécule CRX-527, nous nous sommes particulièrement focalisés sur le rôle du co-récepteur du TLR4, le CD14, dans les réponses innées induites par le CRX-527. Nous avons établi que, de manière inattendue et contrairement à la plupart des ligands TLR4, le CRX-527 induit la production de nombreuses cytokines et chimiokines en complète absence de CD14, même à faible dose. De plus, l'ajout de CD14 sous sa forme soluble ne modifie pas le niveau des réponses associées à la voie de signalisation MyD88 / NF-κB. Cependant, il semblerait que la stimulation de cellules par du CRX-527 en présence de CD14 soluble recombinant, favorise plutôt la voie TRIF / IRF3, comme le suggère l'augmentation du taux de production d'IFNβ et d'activation d'IRF3. La molécule CD14 (membranaire et/ou soluble) ne serait donc pas qu'un simple transporteur de ligands, comme il l'a été décrit par le passé, mais bien une protéine impliquée dans la modulation des réponses induites lors de l'activation du TLR4. Le CD14 jouerait donc un rôle, aussi bien au niveau de la discrimination des ligands, que celle des voies de signalisation activées.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished