Plant platforms for the production of T1D candidate vaccines

Type 1 diabetes (T1D) is an autoimmune disease characterized by the T-cell mediated destruction of insulin-secreting pancreatic β-cells, causing the need of life-long insulin therapy. The autoimmune response is targeted mainly against some proteins present on the β-cells, including the smaller isoform of glutamic acid decarboxylase (GAD65), insulin and the tyrosine phosphate-like IA-2/IA-2B antigen; an autoantigenic role of heat-shock protein 60 (HSP60) released from stressed β-cells has also been demonstrated. Clinical trials are currently testing the possibility of preventing the clinical onset of the disease or delaying the loss of -cell function using immunological strategies based on the induction of tolerance (Tisch & McDevitt, 1994). In this research field, many studies have focused on antigen-specific immunotherapy (ASI), which is based on the delivery of autoantigens or autoantigen-derived peptides at different disease stages and via different routes, resulting in self-tolerance and prevention, delay or suppression of the pathogenic process. ASI is an appealing strategy in autoimmune disease treatment because it selectively inactivates, eliminates, or functionally deviates autoreactive T cells while maintaining the function of the remainder of the immune system (Tisch et al., 1999). The effect is strongly dependent on dose, frequency, route of administration, use of adjuvants, interindividual variations and especially the molecule used for immunization (autoantigen chosen, use of the whole protein or single or mixed peptides) (Peakman et von Herrath, 2010). Given the many potential advantages of this kind of therapy, several studies have been performed by the administration of whole T1D autoantigens or autoantigen peptides, aiming at preventing the disease or suppressing its pathogenic process. In particular, given the promising pre-clinical results obtained in T1D animal model (non obese diabetic -NOD- mouse), human trials based on oral (NCT00419562) and intranasal (NCT00336674) administration of whole insulin are currently underway. A phase II prevention trial based on the use of whole GAD65 (two injections of alum-formulated protein, one month apart) is also in progress in Sweden (NCT01122446). Alternatively, in the last decade many pre-clinical studies in the NOD mice have focused on the use of T1D epitopes from different autoantigens (mainly GAD65, insulin and proinsulin, HSP60). It was demonstrated that the administration of single or mixed T1D-epitopes in the range of µg (usually 40-600) can effectively prevent, delay or suppress the onset of the disease (Arai et al., 2010; Chen et al., 2009; Daniel et Wegmann, 1996; Eldor et al., 2009; Tian et al., 1996; Tisch et al., 1999; Ogino et al., 2000). A modified peptide from HSP60 (DiaPep277) was also tested in humans and two ongoing phase III trials in newly diagnosed T1D patients are assessing its efficacy in preserving β-cell function (NCT00615264 and NCT01103284). The major disadvantage connected with this kind of therapy is the high cost associated with molecule production. In the perspective of ASI therapy for autoimmune treatment the production of immunoreactive molecules in cost-effective recombinant systems would be highly desirable. For this purpose, plant-based systems represent useful platforms for the production of therapeutic proteins, offering advantages in terms of economy and scalability. The aim of this Ph.D. project is the expression of different forms of T1D candidate vaccines in several plant expression platforms, in order to develop high-throughput and cost-effective systems for the production and purification of the target molecules, to use for antigen-specific immunotherapy of T1D. Different forms of GAD65 (one of the major T1D autoantigen) were expressed in several plant-based platforms, demonstrating the feasibility of this approach to obtain high recombinant protein yields. Expression levels and molecular features of the protein obtained in the different plant systems are discussed. Moreover, exploiting the fact that plant viruses can be used as scaffold to display peptides of interest on their surface, plant-viral particles displaying a GAD65 T1D epitope were produced. The efficacy of these molecules as novel candidate vaccine for the prevention of the disease will be tested in NOD mouse animal model.