ABSTRACT

This PhD dissertation has been based on studies performed at Novo Nordisk, Bagsværd. The purpose has been to explore cholera toxin subunit B's (CTB) mechanism of action as an adjuvant for oral tolerance induction in diabetes.

Oral tolerance induction has emerged as an appealing prevention strategy for autoimmune diseases such as type 1 diabetes, multiple sclerosis and rheumatoid arthritis. This approach has already proven its therapeutic efficacy in animal studies, however, recent clinical trials in treatment of autoimmune diseases have been unsuccessful. This could be due to inadequate antigen dosage, intervention late in the course of disease, and/or minute differences in antigenic composition which has previously been demonstrated to impede oral tolerance induction in animals. In animal studies these difficulties are overcome by the conjugation of antigen to cholera toxin subunit B (CTB) which has been used as a carrier-delivery system for antigens in oral tolerance induction. CTB has been the focus of extensive research in the past decades but many aspects of its immunomodulatory action remain unclear. A better understanding of CTB's immunomodulatory actions would allow for more rational drug design in the future.

In order to explore oral tolerance induction in diabetes based on immunological parameters we developed a new, expedient animal model. This model was applied in studies of CTB as an adjuvant for co-administered antigen. We showed that CTB, which has been demonstrated to enhance the tolerogenic potential of coupled antigen, is also an adjuvant for co-administered antigen, reducing antigen dose requirements tenfold. The optimal dose for tolerance induction found using the new model correlated with the optimal dose for preventing diabetes in a virus-induced model. This dose was also found to prevent diabetes in NOD mice with established insulitis.

To clarify CTB's mechanism of action we explored its effect on Ag-specific T cell responses ex vivo which to date have been reported to be inhibitory. We found no enhancement of proliferation by CTB when stimulating cells from mice immunized two weeks earlier. However, CTB strongly enhanced Ag-driven proliferation of cells from mice immunized four weeks earlier. Using Ab depletion and co-culture systems, we showed that CTB directly co-stimulates Ag-specific CD4 + and CD8 + T cell proliferation. ELISPOT assays revealed that CTB enhanced Ag-specific production of IL-2, IL-4 and IFN- γ by four- to fivefold. Studies using CTB as a classical adjuvant confirmed the findings above as CTB mediated a twofold increase in the ex vivo T cell response in a secondary, but not in a primary immunization regimen.

These insights could prove valuable in the design of future vaccine candidates for the therapy of human autoimmune diseases employing CTB as an adjuvant and/or a carrier.