Silkworm Expressed Cholera Toxin B Subunit Fusion Protein Protecting Against Type 1 Diabetes And Alzheimer’s Disease

Type 1 diabetes （TID） and Alzheimer’s disease （AD） are two major diseases threatening human health, and there is a strong relationship between them. Immunotherapy is a potential therapeutic strategy for preventing T1D and AD.Section 1 Oral administration of silkworm-produced GAD65 and insulin bi-autoantigens against type 1 diabetes.TID is an autoimmune diabetes, results from T cell-mediated destruction of insulin-producing β-cells within pancreatic islets. Induction of mucosal tolerance by oral administration of protein antigens is a potential therapeutic strategy for preventing TID; however, the requirement for a large dosage of protein limits clinical applications because of the low efficacy. In this study, we generated a fusion protein CTB-Ins-GAD composed of CTB （cholera toxin B subunit）, insulin, and three copies of GAD65 peptide 531-545, which were efficiently produced in silkworm pupae, to evaluate its protective effect against TID. We demonstrate that oral administration of CTB-Ins-GAD suppressed T1D by up to 78%, which is much more effective than GAD65 single-antigen treatment. Strikingly, CTB-Ins-GAD enhance insulin-and GAD65-specific Th2-like immune responses, which repairs the Thl/Th2 imbalance and increases the number of CD4⁺CD25⁺Foxp3⁺T cell and suppresses insulin-and GAD65-reactive spleen T lymphocyte proliferation and migration. Our results strongly suggest that the combined dual antigens promote the induction of oral tolerance, thus providing an effective and economic immunotherapy against TID in combination with a silkworm bioreactor.Section 2. Oral administration of silkworm-produced Cholera Toxin B Subunit and twofold Amyloid-beta fusion protein against Alzheimer’s Disease.AD is pathologically characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles. Amyloid β-peptide （Aβ） is the main component of senile plaques, and the pathological load of Aβ in the brain has been shown to be a hallmarks of AD. Active and passive immune-mediated cerebral clearance of Aβ has been studied extensively as potential therapeutic strategy. However, immunization with Aβ heighten the risk of meningoencephalitis or micro-hemorrhages. In this study, we generated a CTB-（Aβ42）2 fusion protein composed of CTB （cholera toxin B subunit） and two copies of Aβ42, which were efficiently produced in silkworm pupae, to evaluate its protective effect against AD. We demonstrate that oral administration of CTB-（Aβ42）₂ induced a non-inflammatory Aβ42-specific Th2-like immune responses, and strikingly decreased Aβ levels, area of senile plaques and Tau phosphorylation improved the memory, cognitive function of AD mice, but not significantly changed the levels of L-2、IFN-γ、IL-4 and IL-10. Our results strongly suggest that CTB-（Aβ42）₂ may have therapeutic effects in AD, thus providing an effective and economic immunotherapy against AD in combination with a silkworm bioreactor.