Final Activity Report Summary - IDDM (Development of a novel DNA vaccine and identification of an autoantigen relevant to type I diabetes) Type I diabetes (T1D) is a life-threatening autoimmune disease without cure at present. The disease is caused by the destruction of the insulin-producing beta cells of the pancreas. These cells are attacked by cells belonging to the immune system, mainly by so-called CD4 and CD8 T cells. In this project, we aimed to identify novel autoreactive T cell specificities as well as the generation and test of DNA vaccines for the prevention of type I diabetes using a well-characterised animal model for T1D, the non-obese diabetic (NOD) mouse. To discover new autoreactive T cell specificities, we analysed the autoimmune response against peripherin, an antigen mainly expressed in neuronal tissues but also in beta cells. To this end, we generated two types of reagents, overlapping peptides that cover the entire sequence of peripherin, and MHC class II tetramers that present peptide epitopes to CD4 T cells. MHC class II tetramers are valuable tools to identify peptide-specific CD4 T cells. A total of 14 MHC class II tetramers were generated, including major epitopes with predicted high binding affinities to the MHC class II molecules I-Ag7 expressed by the NOD mouse. Using these reagents we could indeed identify CD4 T cells specific for peripherin in the NOD mouse model. In order to investigate whether additional T cell specificities recognizing peptides different from the ones included in the generated MHC class II tetramers also exist, we are currently using the overlapping peptides in T cell activation assays. These peptides were also used to identify B cell epitopes in the NOD mouse. Using these peptides in ELISAS and dot-blots, we found one major epitope close to the C-terminal end of peripherin which is recognized by a vast selection of B cell hybridomas that had previously been generated from diabetic NOD mice. We will apply this technology to search for autoantibodies in human T1D (at risk) patients. In addition, we generated DNA vaccines to test their efficacy to prevent T1D in the NOD mouse model. We based our approach on the BDC2.5 T cell model, a well characterised CD4 T cell clone that accelerates T1D in the NOD mouse upon transfer. Two DNA vaccines were generated: the first one consists of a mimotope peptide recognised by BDC2.5 that is fused to the C-terminus of lysosomal integral membrane protein II (LIMP II; we have designated this vaccine LIMP II-2.5mi). This vaccine leads to the forced presentation of this peptide in antigen presenting cells by I-Ag7 in the case of the NOD mouse. The second DNA vaccine codes for a fusion protein between CTLA4, the Fc portion of IgG2a, and the BDC2.5 mimotope (CTLA4-Ig-2.5mi). This vaccine was designed to target the 2.5mi peptide towards antigen presenting cells (APCs) in order to improve its presentation to T cells, based on the high affinity of CTLA4 for the B7.1/B7.2 molecules expressed by APCs. We found that treatment of NOD mice with these vaccines could prevent T1D in this animal model. We are currently investigating the mechanism by which T1D is prevented.
