Oral delivery of the intracellular domain of the insulinoma-associated protein 2 (IA-2ic) by bacterium-like particles (BLPs) prevents type 1 diabetes mellitus in NOD mice

Abstract

Antigen-specific immune tolerance, which possesses great potential in preventing or curing type 1 diabetes mellitus (T1DM), can be induced by oral vaccination with T1DM-related autoantigens. However, direct administration of autoantigens via oral route exhibits a low tolerance-inducing effect as a result of the digestion of protein antigens in the gastrointestinal tract (GIT) and therefore, a large dosage of autoantigens may be needed. In this study, bacterium-like particles (BLPs) made from food-grade lactic acid bacteria were used to deliver the intracellular domain of the insulinoma-associated protein 2 (IA-2ic). For this purpose, BLPs-IA-2ic vaccine in which IA-2ic bound to the surface of BLPs was constructed. BLPs enhanced the stability of the delivered IA-2ic based on the stability analysis in vitro. Oral administration of BLPs-IA-2ic significantly reduced T1DM incidence in NOD mice. The mice fed BLPs-IA-2ic exhibited a significant reduction in insulitis and preserved the ability to secrete insulin. Immunologic analysis showed that oral vaccination with BLPs-IA-2ic induced antigen-specific T cell tolerance. The results revealed that the successful induction of immune tolerance was dependent on the immune deviation (in favor of T helper 2 responses) and CD4⁺CD25⁺FoxP3⁺ regulatory T cells. Hence, oral vaccination with BLPs-IA-2ic shows potential for application in preventing T1DM.

Keywords: Type 1 diabetes mellitus; antigen delivery; bacterium-like particles; insulinoma-associated protein 2; oral tolerance.

Figure 1.

Characteristic and identification of recombinant IA-2ic-3LysM fusion protein. (A) Schematic representation of recombinant plasmid pET20b-IA-2ic-3L. (B) Schematic representation of IA-2ic-3LysM fusion protein. Protein anchor (PA) containing 3LysM repeats was fused to the C-terminus of IA-2ic. (C) SDS-PAGE analysis: lane M, protein standard; lane 1, supernatant of IPTG-induced lysate (E. coli BL21 pET20b-IA-2ic-3L) by sonication; lane 2, flow-through of Ni-NTA column; lane 3, elute of Ni-NTA column containing IA-2ic-3LysM. (D) Western blot analysis: lane 1, blank control; lane 2, purified IA-2ic-3LysM fusion protein was detected by Western blotting using anti-His monoclonal antibody.

Figure 2.

Analysis of BLPs-IA-2ic by immunofluorescence microscopy. Immunofluorescence (top) and bright-field microscopy images (bottom) of BLPs alone and BLPs-IA-2ic. Images were acquired at ×100 magnification on an Olympus Fluoview IX70 confocal laser scanning microscope. Scale bar: 5 μm.

Figure 3.

Stability analysis of BLPs-IA-2ic in the stimulated gastric juice. BLPs alone, free IA-2ic-LysM, and BLPs-IA-2ic were incubated with stimulated gastric juice with pH 4.0 (A) and pH 2.0 (B), respectively, and at indicated time, samples were subjected to analysis using ELISA. Data are shown as means ± SD.

Figure 4.

Stability analysis of BLPs-IA-2ic at different temperatures. At –20 °C (A), 4 °C (B) and room temperature (C), BLPs, free IA-2ic-3LysM, and BLPs-IA-2ic were stored for 60 days, and at indicated time, samples were subjected to analysis using ELISA. Data are shown as means ± SD. *p < .05.

Figure 5.

Suppression of diabetes by oral vaccination with BLPs-IA-2ic. Mice were fed BLPs (n = 15), IA-2ic-3LysM (n = 15), or BLPs-IA-2ic (n = 15) once daily during the first week and then three times per week in the following 15 weeks. Diabetes was diagnosed when mouse blood glucose levels were higher than 16 mmol/L for two consecutive weeks as well as it presented with the related symptoms, such as polyuria and weight loss. *p < .05.

Figure 6.

Reduction of insulitis and preservation of C-peptide secretion by oral vaccination with BLPs-IA-2ic. Representative HE staining images of mouse pancreas from BLPs group (A), IA-2ic-3LysM group (B) and BLPs-IA-2ic group (C) at the end of the observation period (40-week-old). Scale bar: 100 μm. (D, E) Comparative analysis of insulitis among groups. All mice alive at the end of the observation period (40-week-old) were tested (n = 4 in BLPs group, n = 6 in IA-2ic-3LysM group, n = 13 in BLPs-IA-2ic group). At least 20 islets per mouse were analyzed. (F) Monitoring of serum C-peptide levels among all groups. For dot plots, each dot represents one mouse. Data are shown as means ± SD. *p < .05.

Figure 7.

Induction of antigen-specific antibodies by oral vaccination with BLPs-IA-2ic. After the final administration (20-week-old), serum samples collected from all mice were quantified for anti-IA-2 antibodies and antibody subtypes. Data are shown as means ± SD. **p < .01.

Figure 8.

Induction of antigen-specific suppression of splenocyte proliferation and a Th1 to Th2 cytokine shift by oral vaccination with BLPs-IA-2ic. At the end of the observation period (40-week-old), all alive mice in each group (n = 4 in BLPs group, n = 6 in IA-2ic-3LysM group, n = 13 in BLPs-IA-2ic group) were euthanized and subjected to splenocyte proliferation test by stimulating with ConA, BSA, and IA-2 (A) and cytokine analysis in the supernatant of splenic cells after IA-2 stimulation (B–F). For each mouse, at least three independent experiments with three or four biological replicates each were performed. For dot plots, each dot represents one mouse. Data are shown as means ± SD. *p < .05, **p < .01.

Figure 9.

Enhancement of Tregs differentiation by oral vaccination with BLPs-IA-2ic. At the end of the observation period (40-week-old), all alive mice in each group (n = 4 in BLPs group, n = 6 in IA-2ic-3LysM group, n = 13 in BLPs-IA-2ic group) were euthanized and subjected to the analysis of the proportion of CD4⁺CD25⁺FoxP3⁺ Tregs in PLNs by flow cytometry (A), and comparison among groups was analyzed (B). For each mouse, at least three independent experiments with three biological replicates each were performed. For dot plots, each dot represents one mouse. Data are shown as means ± SD. *p < .05.