Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 114-127).

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2007. / Committee Chair: Julia Babensee ; Committee Members: Barbara Boyan, John Brash, Andres Garcia, and Niren Murthy. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (M.Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 114-127). Also available in print.

Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

This study investigated the use of non-specific immunostimulants to alleviate stress-mediated suppression of defence mechanisms and subsequent susceptibility to bacterial pathogens in rainbow trout (Oncorhynchus mykiss). One yeast (1-3),)1-6)-β-glucan and a bacterial peptidoglycan were selected as immunostimulants from a panel of test substances on the basis of enhanced intracellular superoxide generation by kidney macrophages stimulated in vitro. Kidney macrophage effector activity was not affected after 1, 2, 3 or 4 weeks of in-feed treatment with 0.05% or 5% of glucan or peptidoglycan. However, production of bactericidal superoxide by inflammatory peritoneal macrophages did increase significantly after four weeks of oral treatment with 0.05% peptidoglycan. Although a single confinement of fish (93% reduction of water volume for five minutes) caused a physiological stress response, as indicated by hyperglycaemia in plasma, kidney and inflammatory macrophage activities were only affected after six daily confinements. Phagocytosis, intracellular superoxide production and killing of Aeromonas salmonicida in vitro by kidney macrophages were significantly reduced. Conversely, production of extracellular superoxide, which may be associated with damage to self, was enhanced. Peritoneal macrophages displayed a similar but less marked respiratory burst response after repeated confinement. Some of the alterations in macrophage function caused by daily confinement were prevented by feeding 0.05% peptidoglycan four weeks before the first confinement. The increase in kidney macrophage extracellular superoxide production caused by repeated confinement was significantly alleviated by in-feed peptidoglycan. Similarly, the decrease in intracellular production by peritoneal macrophages caused by repeated confinement was prevented by in-feed treatment with peptidoglycan. Neither peptidoglycan nor repetitive confinement had any effect on complement lytic activity. These results indicate that dietary peptidoglycan was able to reduce, by regulating macrophage function, the impact of stress on certain bactericidal defences and potential damage to self. However, there was no significant difference in the persistence of viable A. salmonicida in the spleen or blood of infected fish in any of the experimental treatments.

published_or_final_version / Zoology / Master / Master of Philosophy

Coelomocyte migration responses, both random and chemotatic, were examined in the earthworm Lumbricus terrestris. Coelomocyte random migration patterns towards non-stimulatory, non-chemotatic solutions were described. Migration responses to immunostimulatory agents lipopolysaccharides (LPS), N-formly-methionyl-leucyl-phenylalanine (FMLP), sheep erythrocytes, Saccharomyces cerevisiae, Aeromonas hydrophila, Eisenia fetida and Rhabditis pellio were characterized. Chemotaxis was reported to LPS, FMLP, sheep erythrocytes, S. cerivesae and E. fetida. Bio-indicator potential of chemotaxis is discussed relative to variability in migration responses.

Aim: Subunit vaccines have received increasing attention due to their good safety profile. However, subunit vaccines feature low immunogenicity, and soluble antigen is largely ignored by the immune system due to its lack of danger signals. To stimulate an appropriate immune response, subunit antigen vaccines require the addition of an adjuvant and multiple administrations. This study aimed to formulate biodegradable lipid implants, containing a suitable adjuvant, which delivers antigen in a sustained manner. The physico-chemical characteristics of the implants and their ability to stimulate immune responses towards a model antigen in vivo were investigated. Methods: Lipid implants were prepared from phospholipid and cholesterol. Different adjuvants were added, and their potential to induce an immune response to the model antigen ovalbumin (OVA) was investigated. The adjuvants and immunomodulators assessed were Quil-A (QA), imiquimod, and an α-Galactosylceramide (α-GalCer) analogue. Liposomal dispersions were prepared using the lipid film hydration method. These were freeze-dried, and the powder compressed into matrices (diameter of 2 mm). Physico-chemical characterisation was undertaken by transmission electron microscopy (TEM) to investigate the release of colloidal structures (liposomes, immunostimulating complexes [ISCOMs]) upon hydration with release media. Surface changes of the implant matrices were analysed using scanning electron microscopy (SEM). The release of the fluorescently-labelled antigen ovalbumin (FITC-OVA) and its entrapment into the colloidal particles was investigated using spectrofluorophotometry. Additionally, incorporation of the cationic cholesterol derivative DC-cholesterol (DCCHOL) into implants to allow for charge-charge interactions with the negatively-charged OVA, and replacement of the phospholipid with a phospholipid having a higher transition temperature to facilitate the manufacturing process, were attempted and assessed. The immune response stimulated towards OVA released from the implants was analysed in vivo using a C57Bl/6 mouse model. Expansion of CD8⁺ T cells and CD8 T cells specific for the CD8 epitope of OVA (SIINFEKL), as well as expansion of CD4⁺ T cells, were assessed. The ability of implants to stimulate T cell proliferation and interferon-γ production after in vitro restimulation with OVA was analysed. Serum samples were analysed for OVA-specific IgG antibodies. Results: Lipid implants containing Quil-A released colloidal structures upon hydration with buffer. The type of colloids observed by TEM depended on the ratio of QA:cholesterol:phospholipid. Release of OVA was sustained over ten days in implants prepared with egg yolk PC. However, the release kinetics depended strongly on the choice of phospholipid. In vivo, lipid implants containing Quil-A evoked expansion of CD8⁺ T cells. The immune response to one implant was comparable to that obtained by two equivalent injection immunisations. Therefore, the implants obviated the need for multiple immunisations in the vaccination regime tested here. Expansion of CD8⁺ T cells towards the Quil-A-containing implant was greater than that achieved by the immunomodulators imiquimod and the α-GalCer analogue. Quil-A-containing implants produced OVA-specific IgG antibodies to a greater extent than the implants containing imiquimod or α-GalCer. Incorporation of the cationic DCCHOL did not increase the entrapment efficiency of OVA into liposomes. However, the in vivo investigation of DCCHOL-containirig implants showed an adjuvant effect of DCCHOL on antibody responses, but not on cell-mediated immunity. Conclusion: Lipid implants offer great potential as sustained release vaccine delivery systems. The lipid components in the implant formulation were well-tolerated and biodegradable. Lipid implants combine the advantages of sustained release of antigen and particulate delivery by the formation of colloidal particles.

Aim of thesis: Immuno-stimulating complexes (ISCOMs) are particulate vaccine delivery systems composed of Quillaja saponins, cholesterol and phospholipid. ISCOMs are typically spherical cage-like structures with a diameter of 40 nm and carry a negative charge. Incorporation of the respective vaccine antigen into the particles generates more potent vaccines than a simple mixture of both vaccine components. This requires the antigen to display either hydrophobic domains or positive charges, which allow interaction with the ISCOM particles. However, not all antigens fulfil this requirement and modification of these becomes necessary. Hence, the aim of this study was to design novel preparations of ISCOMs with a positive charge, suitable for adsorption of native hydrophilic antigens and poly-nucleotides, and test their potential as a novel vaccine carrier platform. Methods: Two cationic lipids, DC-cholesterol and DOTAP, were selected to prepare the cationic modifications of ISCOMs. DC-cholesterol substituted for cholesterol in classical ISCOMs, whereas DOTAP substituted for their phospholipid component. The phase behaviour of colloidal systems containing Quil-A, phosphatidylcholine (PC) and DC-cholesterol and of colloidal systems comprised of Quil-A, cholesterol and DOTAP was studied by transmission electron microscopy (TEM). Lipid-film hydration was utilised as the first method to prepare these colloidal systems. Selected compositions containing either DC-cholesterol or DOTAP were also prepared by dialysis as second method. A novel third method for preparing homogenous dispersions of classical ISCOMs was developed utilising ethanol injection. This method was also applied in an attempt to prepare cationic modifications of ISCOMs including DC-cholesterol and DOTAP. As in the colloidal systems comprising Quil-A, PC and DC-cholesterol transformations of structures were observed upon dilution with aqueous solutions, these transitions were also studied on classical ISCOMs using TEM and dynamic light scattering techniques. Loading of cationic colloidal structures composed of Quil-A, PC and DC-cholesterol was performed with the model protein antigen ovalbumin (OVA) and a model plasmid, and the resulting structures were analysed by fluorescence spectroscopy, TEM and gel electrophoresis. The immunological properties of non-loaded and OVA-loaded structures were studied in terms of their ability to activate murine bone marrow derived dendritic cells (mBMDC) as antigen presenting cells (APC) and OVA-specific CD8+ T cells in vitro. Results: Substitution of cholesterol in classical ISCOMs with DC-cholesterol resulted in the formation of cationic cage-like structures similar to the classical particles. These were observed in pseudo-ternary Quil-A:PC:DC-cholesterol systems and even in pseudo-binary Quil-A:DC-cholesterol systems prepared by lipid-film hydration. Compositions at which cage-like structures were observed included high weight proportions of DC-cholesterol (> 60%). However, samples were relatively heterogeneous, and aggregation of colloidal structures was observed at equimolar ratios of Quil-A and DC-cholesterol. The ionic strength, pH and composition of the hydration buffer were demonstrated to be important variables influencing the formation of cage-like structures. Morphological changes of pre-formed cationic cage-like structures were observed upon dilution. However, classical anionic ISCOMs showed a similar behaviour. The numbers of cationic cage-like structures appeared to increase upon prolonged storage of samples. Purification of structures and longitudinal analysis of their composition suggested an increased formation of stoichiometrically defined DC-cholesterol:Quil-A:PC complexes over time, rather than a change in composition. The substitution of phospholipid in classical ISCOMs with DOTAP also resulted in heterogeneous dispersions, and aggregation of colloidal structures was observed at equimolar ratios of Quil-A and DOTAP. Phase separation phenomena were proposed based on TEM observations. However, the formation of cage-like particles with a positive [zeta]-potential was not observed. Although ethanol injection was introduced as a novel method to prepare classical ISCOMs, its application did not result in more homogenous dispersions of cationic colloidal structures containing DC-cholesterol or DOTAP. Dialysis also failed to produce higher numbers of well-defined cationic particles, although using this method homogeneous, anionic ISCOM-like particles containing DOTAP were obtained. The efficient adsorption of OVA and plasmid DNA onto cationic structures containing Quil-A, PC and DC-cholesterol was demonstrated. The adsorption process was accompanied with a decrease in [zeta]-potential, aggregation of structures and changes in the ultra-structure, particularly at high protein:lipid ratios. The in vitro immunogenicity of dispersions containing Quil-A, PC and DC-cholesterol was equivalent to that of classical ISCOMs in terms of activation of mBMDC and OVA-specific CD8+ T cells, even though smaller amounts of Quillaja saponins and total lipid were co-delivered with OVA. Furthermore, the uptake of OVA by BMDC appeared to be more efficient in conjunction with the novel cationic dispersions. Conclusions: Cationic colloidal structures containing Quillaja saponins offer great potential as vaccine delivery systems. Their advantages thus far include simple and efficient adsorption of antigen, efficient uptake by APC and immunological activity in vitro. With further development, cationic carriers containing Quillaja saponins may constitute a very potent vaccine delivery platform suitable for a variety of subunit antigens, and suffice both pharmaceutical and immunological requirements.