Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as a Vaccine Adjuvant

Innate immune responses to vaccine adjuvants based on lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls, are driven by Toll-like receptor (TLR) 4 and adaptor proteins including MyD88 and TRIF, leading to the production of inflammatory cytokines, type I interferons, and chemokines. We report here on the characterization of a synthetic hexaacylated lipid A derivative, denoted as glucopyranosyl lipid adjuvant (GLA). We assessed the effects of GLA on murine and human dendritic cells (DC) by combining microarray, mRNA and protein multiplex assays and flow cytometry analyses. We demonstrate that GLA has multifunctional immunomodulatory activity similar to naturally-derived monophosphory lipid A (MPL) on murine DC, including the production of inflammatory cytokines, chemokines, DC maturation and antigen-presenting functions. In contrast, hexaacylated GLA was overall more potent on a molar basis than heterogeneous MPL when tested on human DC and peripheral blood mononuclear cells (PBMC). When administered in vivo, GLA enhanced the immunogenicity of co-administered recombinant antigens, producing strong cell-mediated immunity and a qualitative TH1 response. We conclude that the GLA adjuvant stimulates and directs innate and adaptive immune responses by inducing DC maturation and the concomitant release of pro-inflammatory cytokines and chemokines associated with immune cell trafficking, activities which have important implications for the development of future vaccine adjuvants

Regulation of Immunopathology in Mycobacterium tuberculosis infection

Approximately one third of the world's population is infected with Mycobacterium tuberculosis, which was responsible for about 1.6 million deaths in 2005. In spite of continuing advances in understanding host response to this infection, generation and maintenance of the host immune response remains unclear. In this thesis, we investigate molecules involved in the generation and maintenance of the host response, specifically the granuloma, to M. tuberculosis. We investigated the role of TNF antagonists in reactivation of tuberculosis, and showed that while anti-TNF antibody is superior to TNFR2-Fc fusion molecule in penetrating the granuloma, any blockade of TNF compromises control of acute tuberculosis. We hypothesized that TNF is required for priming T cell responses and that TNF-inducible chemokine receptors function redundantly, allowing one chemokine to compensate in the absence of another. Here, we show that TNF is not required to prime the adaptive immune response, and that TNF-inducible chemokines CXCR3 and CCR5 are simultaneously expendable, refuting the compensation hypothesis in these two chemokines. Reports have implicated unexplored inflammatory molecules in host response to M. tuberculosis infection. We hypothesized that the small chemotactic molecule LTB4 and its receptor BLT1 increase pathology during M. tuberculosis infection. We also hypothesized that osteopontin is required for mediating an effective immune response to tuberculosis by mediating Th1 priming and lymphocyte migration. We show here that neither BLT1 nor osteopontin play a significant role in the inflammatory response to M. tuberculosis. Finally, we investigated the role of ICAM-1 in priming effector and regulatory T cells in response to tuberculosis. We report that ICAM-1 is dispensable for priming and migration of effector T cells, but that ICAM-1 is required for production of inducible Foxp3+ T regulatory cells via TGFâ1 stimulation. We hypothesize that the reduction in T regulatory cells exacerbates the immune response, allowing greater inflammation in the lungs, potentially causing overwhelming inflammation. This body of work contributes to the understanding of the host response to tuberculosis by investigating activity of cytokines, chemotactic molecules and adhesion molecules in balancing the host response to M. tuberculosis infection

Aberrant TGF-β signaling reduces T regulatory cells in ICAM-1-deficient mice, increasing the inflammatory response to Mycobacterium tuberculosis

Foxp3+ T regulatory cells are required to prevent autoimmune disease, but also prevent clearance of some chronic infections. While natural T regulatory cells are produced in the thymus, TGF-β1 signaling combined with T-cell receptor signaling induces the expression of Foxp3 in CD4+ T cells in the periphery. We found that ICAM-1−/− mice have fewer T regulatory cells in the periphery than WT controls, due to a role for ICAM-1 in induction of Foxp3 expression in response to TGF-β1. Further investigation revealed a functional deficiency in the TGF-β1-induced translocation of phosphorylated Smad3 from the cytoplasmic compartment to the nucleus in ICAM-1-deficient mice. This impairment in the TGF-β1 signaling pathway is most likely responsible for the decrease in T regulatory cell induction in the absence of ICAM-1. We hypothesized that in the presence of an inflammatory response, reduced production of inducible T regulatory cells would be evident in ICAM-1−/− mice. Indeed, following Mycobacterium tuberculosis infection, ICAM-1−/− mice had a pronounced reduction in T regulatory cells in the lungs compared with control mice. Consequently, the effector T-cell response and inflammation were greater in the lungs of ICAM-1−/− mice, resulting in morbidity due to overwhelming pathology