Theses and Dissertations at Montana State University (MSU)
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Item Low dose tolerance vaccine platform, reovirus protein sigma 1 and treatment of autoimmunity(Montana State University - Bozeman, College of Agriculture, 2008) Rynda, Agnieszka; Chairperson, Graduate Committee: David Pascual.Effective treatments for multiple sclerosis (MS) are problematic due to its unknown etiology. Experimental autoimmune encephalomyelitis (EAE) in rodents mimics MS. Mucosal treatment of EAE with antigens to induce tolerance is effective, but requires large and/or multiple administrations, which introduces an allergy risk. We utilized reovirus adhesin, protein sigma 1 (p sigma1), to improve mucosal auto-antigen delivery and show that a single low-dose of p sigma1-based vaccines induces tolerance and prevents autoimmunity when administered nasally. We engineered three p sigma1-based vaccines carrying chicken ovalbumin (OVA-p sigma1) and/or myelin antigens (PLP:OVA-p sigma1, MOG-p sigma1). When mice were nasally immunized with OVA-p sigma1, tolerance to OVA was established. This tolerance resisted co-administration of mucosal adjuvants and peripheral challenge with OVA. P sigma1-mediated tolerance relied upon specific IL-10- producing regulatory T (T reg) cells, which inhibited OVA-specific CD4+ T cell proliferation. OVA-p sigma1 did not generate tolerance in IL-10-deficient mice presumably by a failure to induce T reg cells. Mucosal, but not systemic p sigma1 delivery, induced tolerance, while mice lacking mucosal inductive tissues were resistant to p sigma1-mediated tolerance. Likewise, PLP:OVA-p sigma1 and MOG-p sigma1 protected mice against relapsing-remitting or acute EAE, respectively. Protection against PLP139-151-induced EAE was accomplished by PLP:OVA-p sigma1, but not OVA-p sigma1, implicating antigen-specificity of p sigma1-mediated tolerance. Moreover, MOG-p sigma1, but not PLP:OVA-p sigma1, ameliorated MOG35-55-induced EAE via apoptosis of encephalitogenic CD4+ T cells. The PLP:OVA-p sigma1- or MOG-p sigma1-mediated protection against EAE depends on specific IL-10+ T reg cells and is supported by IL-4+ Th2-type cells. Adoptive transfer of PLP:OVA-p sigma1-primed T reg cells entirely prevented EAE development in mice; however, transfer of PLP:OVA-p sigma1-specific CD25- CD4+ Th2 cells significantly reduced and delayed clinical EAE. Aggressive EAE, due to the TGF-beta which induced activation of Th17 cells, was observed in mice dosed with PLP:OVA-p sigma1 and were functionally depleted of T reg cells. Concomitant inactivation of TGF-beta and T reg cells induced Th2 cells bias and re-established PLP:OVA-p sigma1-mediated protection against EAE. IL-10-producing B cells supported MOG-p sigma1-mediated protection against EAE, as MOG-p sigma1-dosed B cell-deficient mice developed attenuated disease. Adoptive transfer of T reg cells, but not Th2 or B cells from MOG-p sigma1-dosed B6 mice to diseased IL-10-/- mice, significantly accelerated recovery from EAE. These data demonstrate the feasibility of using p sigma1-based single-dose delivery system to prevent and/or treat autoimmunity.Item Vaccine platform for infection or autoimmune diseases using an ETEC fimbrial scaffold(Montana State University - Bozeman, College of Agriculture, 2009) Jun, SangMu; Chairperson, Graduate Committee: David Pascual.The expression of enterotoxigenic Escherichia coli (ETEC) fimbriae (colonization factor antigen I (CFA/I) or K99) on the surface of a Salmonella vaccine vector confers protection against ETEC challenge. Application of such fimbriae as a treatment for the proinflammatory disease, experimental autoimmune encephalomyelitis (EAE), or as a molecular scaffold for heterologous antigen expression by cloning enterohemorrhagic E. coli (EHEC) LPS peptide mimetics into the K99 fimbriae to produce a dual vaccine for ETEC/EHEC was investigated. The expression of CFA/I fimbriae by a Salmonella vaccine vector stimulates a biphasic T helper (Th) cell response and suppresses proinflammatory responses suggesting that CFA/I fimbriae may be protective against proinflammatory diseases. To test this hypothesis, SJL/J mice were vaccinated with Salmonella-CFA/I vaccine 1 or 4 wks prior to induction of EAE induced with encephalitogenic proteolipid protein (PLP) peptide, PLP₁₃₉-₁₅₁. Mice receiving Salmonella-CFA/I vaccine recovered completely from the mild acute clinical disease and showed only mild inflammatory infiltrates in the spinal cord. This protective effect was accompanied by a loss of encephalitogenic IFN-gamma secreting Th1 cells and replaced with increases in IL-4-, IL-10-, and IL-13- producing Th2 cells. These data suggest that Salmonella-CFA/I is an anti-inflammatory vaccine capable of suppressing proinflammatory cells to protect against EAE via immune deviation. To obtain an effective nasal vaccine for ETEC, the fanC gene of ETEC K99 major structural gene was cloned onto the reovirus adhesin, protein sigma1, which has been shown as an M cell targeting molecule. Although FanC/protein sigma1 fusion protein was successfully expressed, this vaccine failed to elicit immune responses against native FanC protein, presumably because of improper protein folding. Using K99 fimbriae as a molecular scaffold, a LPS peptide mimetic for EHEC was cloned into the fanH gene of K99 fimbriae minor structural gene to enable multiple antigenic peptide expression, resulting in an ETEC/EHEC dual vaccine. Insertion of peptide mimetic into fanH gene had no adverse effect on the formation of polymerized K99 fimbriae. However, various oral immunization regimens failed to induce the protective secretory IgA responses against the LPS mimetic peptide, although serum IgG antibodies were induced.