Abstract
Induction of mucosal tolerance by oral administration of protein antigens is a potential therapeutic strategy for preventing and treating type 1 diabetes (T1D); 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 T1D. 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 Th1/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 T1D in combination with a silkworm bioreactor.
Highlights
Oral tolerance refers to the physiological response of an organism remaining in a state of specific immunological unresponsiveness to orally delivered antigens [1, 2]
A flexible hinge tetrapeptide (GPGP) was introduced among the three peptides to maintain the natural conformation of insulin, GAD65, and cholera toxin B subunit (CTB)
Our results showed that mice treated with CTB-Ins-glutamic acid decarboxylase (GAD) produced a higher proportion of CD4+CD25+Foxp3+ T cells in the pancreas and pancreatic lymph nodes (PLN) CD4+ T cell populations compared with the CTB-GAD group (21.23±0.64% vs 15.14±0.71% in the pancreas; 15.32±0.59% vs. 10.81±0.51% in the PLN) (Fig 6A and 6B)
Summary
Oral tolerance refers to the physiological response of an organism remaining in a state of specific immunological unresponsiveness to orally delivered antigens [1, 2]. Type 1 diabetes (T1D) is a spontaneous organ-specific autoimmune disease [3,4,5], and a series of autoantigens have been identified in both humans [6, 7] and NOD mice [8], including insulin [6] and glutamic acid decarboxylase (GAD) [8] Oral administration of these antigens has shown efficacy in preventing T1D in non-obese diabetic (NOD) mice [9,10,11,12,13,14,15]. Subgroup analyses revealed that oral insulin delayed diabetes onset for up to 5 years in patients who had high insulin autoantibody levels Based on these subgroup results, Type 1 Diabetes TrialNet is conducting a new oral insulin prevention trial to verify or refute this observation [20] (www.clinicaltrials.gov/ct2/show/NCT00419562).
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