Date of Award

2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Ghosh, Swapan

Abstract

Adjuvants are used widely in vaccine formulations. However for humans, choices are very limited. Since they are selected empirically, it is not expected that any two adjuvants would influence immune mechanisms the same way. However they all influence host microenvironment, antigen presentation, and retention of immunological memory. This study focuses on new terpenoid adjuvants based on phytol derivatives. We previously observed that phytol and one of its derivatives PHIS-01 (a phytol-based immunostimulant, phytanol) are excellent adjuvants. To gain an understanding of the structural features important for adjuvanticity, we further studied compounds derived from a diterpene Phytol. We designed two new phytol derivatives, PHIS-02 and PHIS-03 (aminated and mannosylated compounds respectively). In this study we investigated their relative safety and efficacy compared to PHIS- 01 (phytanol) and other commonly used adjuvants that include alum, Freunds' adjuvants and SIS (extra-cellular matrix). In addition, we examined how changes at the polar terminus affect adjuvanticity of PHIS-01, PHIS-02, PHIS-03 in term of host microenvironment and safety profile. Using these adjuvants as emulsions with different soluble protein antigens, ovalbumin and a hapten-protein conjugate phthalate-KLH, we evaluated in both autoimmune resistant and susceptible murine models. The following immunological parameters were studied: 1) effects on antibody responses in terms of titers, specificities and isotypic profiles; 2) effects on T-helper cells, cytokines, and chemokines milieu; 3) involvements of apoptotic and/or necrotic activity and inflammasome pathways as their primary modes of action. Our results indicate that: 1)modified phytol-derived adjuvants significantly augment antibody response of isotypes IgG1 and IgG2a, promote effective T cell proliferation and exhibit no adverse autoimmune anti-DNA response in either autoimmune or non autoimmune mice. 2) Phytol derivatives function by activation of antigen-presenting cells involving apoptotic/necrotic effects on target cells. 3) Phytol derivatives improve vaccine immunogenicity by promoting regulated and nonpathogenic inflammatory changes in the immediate microenvironments, as characterized by mobilization of chemo tactic factors (MCP-1, KC, MIP-1, LIX, lymphotactin, eotaxin), growth factors (MCSF, GCSF, GM-CSF), and cytokines that mobilize innate and adaptive immunity and lead to T helper polarization and a magnified antibody response 4) PHIS -01, compared to PHIS-03 and alum , is a better activator of genes in the inflammasome pathways. In conclusion, our findings also clearly highlight the importance of bonds and functional moieties in shaping the adjuvanticity of phytol derivatives. Hydrogenation of phytol generates PHIS-01 which is a very safe and superior adjuvant in terms of the quality and magnitude of the overall immune response evoked. However, modification of its polar terminus of PHIS-01 with a hydrophilic mannose moiety (PHIS-03) profoundly changes the cytokine/chemokine milieu and favors T-helper type 2 rather than the T-helper type1 induced by PHIS-01.

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