Despite the progress in elucidating antigens for both therapeutic and prophylactic vaccines, safety concerns over current vaccine delivery vehicles and adjuvants has limited the development of new vaccines. In particular, there is an urgent need for effective vaccines capable of stimulating cytotoxic T lymphocyte (CTL) responses against intracellular pathogens or tumor cells. Liposomes are under investigation as a particulate vaccine delivery system with the required safety profile and demonstrated ability to target antigens to dendritic cells (DC), the cells of the immune system responsible for initiating effective and long lasting CTL immune responses. Unmodified liposomes however, are inherently non-immunogenic and thus not capable of stimulating activation of DC, which is a necessary step in immune activation. In this thesis the use of modified liposomes to more efficiently target vaccine antigens to DC and then activate the DC sufficiently to initiate down-stream immune responses was investigated. In the first approach to liposome modification, mannosylated phospholipids were incorporated within the liposome bilayer to target C-type lectins on DC. Incorporation of mono- or tri-mannosylated phospholipids within liposomes was found to be an effective means of attaching mannose-containing ligands to the liposome surface without compromising the integrity of the liposome structure. The uptake of tri-mannose-containing liposomes was enhanced in human monocyte derived DC (MoDC) compared to both unmodified liposomes and mono-mannose-containing liposomes. In contrast, neither mono- nor tri-mannose-containing liposomes were taken up by murine bone marrow derived DC (BMDC) to a greater extent than unmodified liposomes. This finding may reflect the differences in ligand specificity for C-type lectins on DC derived from different mammalian species. It was also found in these studies that increased uptake of liposomal antigens by DC does not necessarily result in increased DC activation, as evidenced by a lack of up-regulation of DC surface activation markers and ability to stimulate T cell proliferation. The second approach to liposome modification involved the incorporation of lipid core peptides (LCPs) into the liposome structure. LCPs alone were demonstrated to be able to stimulate DC and subsequent CD8+ T cell activation in vitro. LCP-based vaccines were also able to stimulate effective cytotoxic immune responses in vivo, and protect against tumor challenge, but only if administered in alum with CD4 help. Liposomes containing LCPs were able to stimulate greater DC activation and subsequent CD8+ T cell proliferation in vitro compared with unmodified liposomes. In the in vivo studies however, LCP-containing liposomes were not able to stimulate a cytotoxic immune response or protect against tumor challenge as effectively as LCP administered in alum. In the final approach to liposome modification, inclusion of the adjuvant Quil A was investigated for its ability to increase the immunogenicity of LCP-containing liposomes. It was found that small amounts of Quil A could be incorporated into liposomes without compromising the liposome bilayer. The inclusion of as little as 2% Quil A was able to stimulate DC activation and subsequent T cell proliferation in in vitro studies. In addition, immunisation of mice with LCP-containing liposomes with incorporated Quil A was found to stimulate an in vivo CTL immune response comparable to LCPs administered under optimal vaccine conditions. In conclusion, the work presented in this thesis demonstrates that modified liposomes are a useful vaccine delivery system for the initiation of in vivo cytotoxic and prophylactic immune responses.

Thesis (Ph. D.)--Lehigh University, 2000. / Includes vita. Includes bibliographical references (leaves 288-301).

Probiotics are living microorganisms, which when administered in adequate amounts confer a health benefit on the host. They have been reported to relieve acute diarrhoea, atopic dermatitis and irritable bowel syndrome in disease-specific animal studies and in human intervention trials. However, probiotics are regularly consumed by general healthy population with limited knowledge in the immunomodulation of probiotics of local and systemic immune responses in healthy experimental models. Serving as the first line of defense against microbial infections and the largest immunological organ in animal host, the epithelium lining the small and large intestine is supposed to be the first organ to encounter probiotics as probiotics are always orally taken. It is believed that probiotics regulate the local immunities in the gut, which acts as the pivot in modulating the systemic immune responses. Accordingly, it was hypothesized that probiotic bacteria can modulate both local and systemic immune responses in healthy population; and the immunomodulation of combination of probiotics is different from that of individual strains. Wildtype healthy C57BL/6 mice were fed with different probiotic strains − Lactobacillus rhamnosus GG (LGG), Lactobacillus rhamnosus LC705 (LC705), Bifidobacterium breve Bb99 (Bb99), Propionibacterium freudenreichii ssp. shermanii JS (PJS) or Escherichia coli Nissle 1917 (EcN), or mixture of probiotics − GGmix (LGG, LC705, Bb99 and PJS) and ECPJSmix (PJS and EcN), for three weeks. After that, intestine, liver, spleen and blood were investigated. Probiotics suppressed intestinal T helper (Th)17 immune response but enhanced systemic (hepatic and splenic) Th17 immune response, suggesting that immune homeostasis was maintained in healthy individuals. Mechanism of action of LGG was further studied in this project as LGG is the widely studied probiotics. It was hypothesized that LGG exerts immunomodulatory effects by bacteria cells and/or its derived soluble factors such as lactic acid. Immunomodulatory effects of LGG cells and their soluble factors on dendritic cells (DCs), macrophages and monocytes from healthy blood donors were investigated as antigen-presenting cells (APCs) are pivots of bridging innate and adaptive immunities. Cytokine secretion profile, expressions of toll-like receptors (TLRs) and activation-related receptors of the APCs were examined. Both LGG cells and their soluble factors promoted type 1-responsiveness while soluble factors promoted type 17-responsiveness as well. Yet, lactic acid seemed not to be the one which enhanced type 1 and type 17 immune responses in soluble factors. With better understanding on the immunomodulation of probiotics in healthy models, prophylactic efficacy of probiotics in preventing infections and diseases can be availed. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Thesis (Honors)--College of William and Mary, 2009. / Includes bibliographical references (leaves 62-68). Also available via the World Wide Web.

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy