DERIVATION OF TOLEROGENIC THERAPEUTIC VACCINES FOR MOUSE MODELS OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

Abstract

Multiple sclerosis (MS) is a chronic inflammatory disease that results in autoimmune demyelination of the human central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is a widely-studied animal model of multiple sclerosis which reflects many of the histopathological and immunological features of MS. Because currently used therapies for multiple sclerosis result in broad-spectrum immunosuppression, antigen specific vaccines may be an important alternative. Whereas the concept of traditional anti-microbial vaccination is to introduce the immune system with the foreign microbes to develop memory T and B cells, self-reactive T cell suppression is the fundamental principle of tolerogenic vaccines. For EAE, contemporary research is focused on the development of potent tolerogens. Fusion proteins of cytokine and neuroantigen (NAg) are assumed to be such potent tolerogens. Antigen presenting cells (APC) are targeted by the fusion proteins in a cytokine dependent manner. These APC have been shown to play a fundamental role in inducing sustainable self-tolerance. That is, autoimmune effector T cells are eliminated by regulatory T (Treg) cells that are stimulated and expanded in a negative thymic selection process where dendritic cells (DC) play an inevitable role. The principle is that the cytokine domain of the fusion protein will interact with cytokine receptors on the APC and facilitate the presentation of covalently linked NAg to self reactive T cells and thereby, induce a co-inhibitory signal leading to cytotoxicity and killing of NAg-specific autoreactive T cells. In an approach to induce tolerogenic response in EAE, I have studied two fusion proteins consisting of interferon-beta (IFN-[beta]) fused to myelin oligodendrocyte glycoprotein (MOG) and IFN-[beta] fused to proteolipid peptide (PLP). Both fusion proteins were potent tolerogens. The fusion genes were cloned in pIRES2 expression plasmids and then expressed stably in human embryonic kidney cells, and the fusion proteins were purified by affinity chromatography. Functional integrity of the IFN-[beta] domain within the fusion protein was confirmed by a T cell anti-proliferative assay and a class-I MHC induction assay. Biological activity of NAg domains were tested by NAg specific T cell proliferative assays. The tolerogenic effect of the fusion protein was assessed in two ways. First, the preventive role of IFN-[beta]-PLP was examined by administering the protein before EAE induction which inhibited the subsequent incidence of EAE. Second, IFN-[beta]-MOG was administered after disease onset which halted disease progression. In both cases, covalent linkage between the IFN-[beta] and NAg domain was required to restore the tolerance.