archives@tulane.edu / Infectious diseases are one of the leading causes of morbidity and mortality in humans. Vaccines are one of the best tools to protect against pathogens; however, there remains room for improvement in vaccine design. In particular, different adjuvants can be used to improve or alter the efficacy of vaccines. We hypothesize that adjuvant choice can be used to specifically modify the antigen- specific B cell response and target the response to mucosal tissues. Using an Ova- specific B cell tetramer to track the B cell response, we discovered that the adjuvant double mutant heat labile toxin (dmLT) from E. coli induced the greatest proliferation and activation of Ova-specific B cells two weeks after a single intramuscular or intradermal injection when compared to Ova with no adjuvant. Interestingly, anti-Ova IgG titers plateaued five weeks after the intradermal injection but was still increasing eight weeks after an intramuscular injection. Consistent with previous T cell data, dmLT was able to provoke antigen-specific B cells to express the mucosal homing marker a4b7 and induced residence of vaccine-specific B cells in the Peyer’s patches, lamina propria of large intestines, and lungs. This property was unique to dmLT as the adjuvant CpG was not able to induce similar tissue residence of Ova-specific B cells. Additionally, when dmLT was compared to the aluminum salt-based adjuvant (alum), that is used in the most vaccines, dmLT is able to enhance the antigen-specific B cell response more than alum regardless of route or quantity of injections. We also determined that dmLT produced a higher level of anti-Ova IgG compared to alum. Finally, we showed that combining dmLT with another adjuvant, monophosphoryl lipid A (MPL-A), improved proliferation and activation of antigen-specific B cells and generated higher amounts of Ova-specific IgG compared to any of the single adjuvants alone. Interestingly, while dmLT was a more effective at activating B cells, MPL-A induced higher titers of antibodies than dmLT. These studies present the first time that antigen-specific B cells have been found in mucosal tissues following parenteral immunization as well as provide insight into the effects that combining adjuvants have on B cells. / 1 / John Timothy Prior

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 4 uppsatser.

No description available.

This paper will attempt to determine whether the increased C'4 previously shown following antigen and adjuvant injection could be correlated to antibody increase following antigen injection.

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.

Immunostimulatory oligodeoxynucleotides (ODN) containing unmethylated CpG motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through the triggering of the endosomally localized TLR9 by a poorly understood mechanism. The immunopotency and broad range of activity of CpG ODN makes it a promising immunotherapeutic for the treatment and prevention of cancer and other diseases. However, rapid degradation of ODN by serum nucleases, low levels of accumulation in target tissue and lack of specificity for and poor uptake into target cells following systemic administration pose significant hurdles for the clinical application of CpG ODN. This thesis describes the immunostimulatory properties of CpG ODN encapsulated in liposomal nanoparticles (LN), a delivery system that overcomes many of the problems impeding the clinical development of "free" ODN. In particular, it is shown that LN delivery of CpG ODN specifically targets the ODN for uptake by immune cells in vivo, providing a basis for significantly enhanced immunostimulatory activity, including more potent innate and adaptive immune responses, that ultimately improve anti-tumour efficacy. A particular focus of this thesis concerns previous observations that methylated sequences in ODN (mCpG ODN) are immunologically inert. It is shown that encapsulation of mCpG ODN in LN results in immunostimulatory activity that is equal to or greater than that observed for LN formulations of the equivalent unmethylated form as judged by various immune parameters and anti-tumour efficacy. Further, it is shown that both LN-mCpG ODN and LN-CpG ODN exert their immunostimulatory effects via TLR9 based on preliminary in vitro results and confirmed by studies performed in TLR9 knockout animals. The mechanisms responsible for the differentiation between both CpG ODN and mCpGODN and how encapsulation endows immunostimulatory potential are explored. It is shown that discrimination occurs upstream of TLR9 and that the lack of immunological activity of free mCpG ODN is not due to differences in uptake, trafficking to endosomal compartments or ability to bind to TLR9, when compared with CpG ODN, but rather due to its ability to colocalize with TLR9 in the endosomal compartment. It is proposed that whereas the uptake of free CpG ODN results in the induction of the Src Family Kinase signalling cascade which mediates the migration of TLR9 from the ER to the late endosome, the uptake of free mCpGODN does not. However, it is suggested that encapsulation bypasses the methylation specific recognition of CpG ODN, allowing for the activation of SFK signalling resulting in subsequent co-localization of TLR9 and mCpG ODN in the endosome thus initiating immunostimulatory activity.

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

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 126-161).

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