Silkworm-expressed Cholera Toxin B Subunit-insulin/insulin B Chain Fusion Protein Against Autoimmune Diabetes

Type 1 diabetes is unquestionably an autoimmune disease, as evidenced by the presence of β-cell-reactive autoantibodies and T lymphocytes;T cell-mediated transfer of the disease in non-diabetic mice, rats, and humans;and sensitivity of the disease to immunosuppressive therapy. T cells are predominantly, if not exclusively, involved in creating the islet lesions that lead to β-cell atrophy after a stage of reversible inflammation. In the development of the disease, insulin (INS) or its precursors, glutamic acid decarboxylase (GAD) and heat shock protein 60 (HSP60), are the primary autoantigens recognized by the T cells.1. Oral administration of a cholera toxin B subunit-insulin fusion protein produced in silkworm protects against autoimmune diabetesThe oral administration of disease-specific autoantigens can induce oral immune tolerance and prevent or delay the onset of autoimmune disease symptoms. It is critical to obtain enough autoantigen to successfully induce oral tolerance. The cholera toxin B subunit (CTB) has been used to overcome such limitations by serving as a mucosal carrier molecule for chemically or genetically conjugated autoantigens for the induction of oral tolerance. The therapeutic applications of CTB-mediated oral tolerance may be a potential therapeutic strategy for preventing and treating autoimmune diseases including type I diabetes.Here we describe the construction of an edible vaccine consisting of a 20-kDa fusion protein composed of CTB and insulin that is produced in silkworm larvae at levels of up to 0.3 mg per ml of hemolymph. The silkworm bioreactor produced this fusion protein vaccine as the pentameric CTB-insulin form, which retained the GMl-ganglioside binding affinity and the native antigenicity of CTB and insulin. Non-obese diabetic mice fed hemolymph containing microgram quantities of the CTB-insulin fusion protein showed a prominent reduction in pancreatic islet inflammation and a delay in the development of symptoms of clinical diabetes. Theseresults demonstrate that the silkworm bioreactor is a feasible production and delivery system for an oral protein vaccine designed to develop immunological tolerance against T cell-mediated autoimmune diabetes by regulatory T cell induction. This study offers a new approach for preventing and treating of type I diabetes by using the silkworm resources to develop oral vaccine.2. Incorporation of partial polyhedrin homology sequences (PPHS) enhances the production of cloned foreign genes in a baculovirus expression systemBaculovirus expression vector systems (BEVS) have been used extensively for high-level expression of cloned foreign genes. In many instances, the levels of recombinant protein(s) produced in insect cells and larvae are insufficient for experimental purposes. Thus, new techniques and methods are needed to increase significantly the protein expression levels in BEVS. In this report, we describe the incorporation of a 15-bp element derived from the 5'-end partial sequence of the polyhedrin gene, which contains the non-coding sequence ATAAAT and the coding sequence ATGCCGAAT, into the 5'-end of the CTB-insulin (CTB-INS) fusion gene. With the addition of the partial polyhedrin homology sequences (PPHS), two extra amino acids (PN) were added to the N-terminus of the modified CTB-INS fusion protein (mCTB-INS). This new fusion protein was expressed in both insect cells and larvae using BEVS. We found that the addition of PPHS enhanced four-fold the expression of CTB-INS both in insect cells and larvae. Further analysis revealed that the additional two amino acids in mCTB-INS did not significantly affect binding affinity for GM1 ganglioside. Therefore, the PPHS can be used as a constitutive element immediately downstream of the polyhedrin promoter to induce significant increases in the expression levels of cloned foreign genes.3. Prevention of diabetes in non-obese diabetic (NOD) mice by oral administration of a cholera toxin B subunit-insulin B chain fusion protein expressed in silkwormThe insulin B-chain was chosen as it has been shown to contain the predominantepitope of insulin that is implicated in the development of autoimmune diabetes in animal models, and feeding of epitopes from the B-chain can be used to prevent the development of disease in a manner similar to the whole protein.In this report, we describe the construction of an edible vaccine consisting of a 17-kDa fusion protein composed of CTB and insulin B chain (InsB) that is produced in silkworm larvae at levels of up to 0.97 mg/ml of hemolymph. The silkworm bioreactor produced this fusion protein vaccine as the pentameric CTB-InsB form, which retained the GMl-ganglioside binding affinity and the native antigenicity of CTB. Non-obese diabetic mice fed middle-dose of hemolymph containing microgram quantities of the CTB-InsB fusion protein showed a prominent reduction in pancreatic islet inflammation and a delay in the development of symptoms of clinical diabetes. This study demonstrates that silkworm-derived CTB-InsB fusion protein can be used as an ideal oral protein vaccine for suppression of insulitis in NOD mice.