Bacterial and Plant Enterotoxin B Subunit–Autoantigen Fusion Proteins Suppress Diabetes Insulitis

Abstract

Several bacterial and plant enterotoxin B subunit-islet autoantigen fusion proteins were compared for their ability to serve as islet autoantigen carriers and adjuvants for reduction of pancreatic islet inflammation associated with type 1 diabetes. The cholera toxin B subunit (CTB), the heat-labile toxin B subunit from enterotoxigenic Escherichia coli (LTB), the Shigella toxin B subunit (STB), and the plant toxin ricin B subunit (RTB) were genetically linked to the islet autoantigens proinsulin (INS) and glutamic acid decarboxylase (GAD). The adjuvant-autoantigen gene fusions were transferred to a bacterial expression vector and the corresponding fusion proteins synthesized in E. coli. The purified adjuvant-autoantigen proteins were fed to 5-wk-old nonobese diabetic (NOD) mice once a week for 4 wk. Histological examination of pancreatic islets isolated from inoculated mice showed significant levels of insulitis reduction in comparison with uninoculated mice. The ratio of serum anti-INS and anti-GAD IgG2c to IgG1 antibody isotype titers increased in all ligand-autoantigen inoculated animal groups, suggesting an increase in effector Th2 lymphocytes in B subunit-mediated insulitis suppression. The results of these experiments indicate that bacterial and plant enterotoxin B subunit ligand-autoantigens enhance insulitis reduction in NOD mice. This research prompts further exploration of a multiadjuvant/autoantigen co-delivery strategy that may facilitate type 1 diabetes prevention and suppression in animals and humans.

Expression and Characterization of Synthetic Heat-Labile Enterotoxin B Subunit and Hemagglutinin–Neuraminidase-Neutralizing Epitope Fusion Protein in Escherichia coli and Tobacco Chloroplasts

“…These results indicate that recombinant LTB and LTB-HNE exist in E. coli as native conformations, which follows from their ability to form pentamers under non-heating conditions and without DTT. This finding is supported by other studies, in which a C-terminal extension of LTB did not affect pentamer formation or its export to the periplasm (Green et al 1996;Carter et al 2006). On the other hand, both LTB and LTB-HNE in bacterial cells transformed with pET21a, which does not contain a signal sequence, were overexpressed as seen in the total cell pellet and remained in the spheroplast as insoluble inclusion bodies (data not shown).…”

“…These results suggest that ETEC heat-labile enterotoxin B subunit (LTB) could be used as the potential adjuvant for foreign antigens without the toxicity of heat-labile enterotoxin A subunit. Furthermore, several LTB-antigen fusion proteins have been reported to show immunogenicity that induced Th1/Th2 lymphocytes in animal experiments (Carter et al 2006) and upregulated MHC class II peptides (Nashar et al 2001;Bone et al 2002). Based on this research, the development of a subunit vaccine utilizing an HN antigen or peptides inducing neutralizing antibodies and the co-administration of fusion-type proteins with LTB as a mucosal adjuvant might effectively induce the neutralizing antibody against NDV.…”

Extracellular, cell-permeable survivin inhibits apoptosis while promoting proliferative and metastatic potential

“…Recombinant transferrin protein was purchased from Sigma-Aldrich. The expression vector PRSET-CTB contained a gene encoding the entire cholera toxin B subunit (CTB) protein (11.6 kDa), driven by the T7 bacteriophage promoter region and containing an oligonucleotide encoding 6 histidines immediately upstream of the CTB gene for nickel column isolation of the recombinant protein was built as described (Carter et al, 2006). Briefly, CTB protein synthesis was stimulated by addition of 90 mg IPTG to the bacterial culture for 6 h at 371C.…”

Antigen-Specific Therapeutic Approaches in Type 1 Diabetes

“…Administration of GAD through other routes, including intrathymic (Tisch et al 1993;Cetkovic-Cvrlje et al 1997), intraperitoneal (Pleau et al 1995), intranasal (Tian et al 1996), or oral routes (in the form of GAD67- or GAD65/IL-4-transgenic plant tissues [Ma et al 2004]) also had antidiabetogenic effects. Rather than inducing classical forms of T-cell tolerance, some of these approaches induced GAD-specific Th2 responses, elevating serum IgG1 levels while decreasing GAD-specific IFNg production (Tian et al 1996;Ma et al 1997;Carter et al 2006). Whether these antigen-specific Th2 responses were directly responsible for disease protection, however, was not formally established.…”