ABSTRACT
The B subunit of Escherichia coli heat-labile enterotoxin (LTB) acts as efficient mucosal carrier for conjugated antigens. We expressed two heterologous proteins using E. coli as a host: a hybrid consisting of LTB and the A, B and C domain of synapsin (LTBABC) and the separated ABC peptide of this synaptic protein. Refolded LTBABC and LTB bound to the GM1 receptor and internalized into CHO-K1(GM1+) cells. LTBABC showed enhanced solubility and cell binding ability respect to the former hybrid LTBSC. Several oral doses of LTBABC were administered to rats with experimental autoimmune encephalomyelitis (EAE) from induction to the acute stage of the disease. This treatment decreased disease severity, delayed type hypersensitivity reaction and lymph node cell proliferation stimulated by myelin basic protein. Amelioration of EAE was also associated with modulation of the Th1/Th2 cytokine ratio, increased TGF-ß secretion in mesenteric lymph nodes as well as expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cell population. These results indicate that the fusion protein LTBABC is suitable for further exploration of its therapeutic effect on EAE development.
Subject(s)
Bacterial Toxins/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Enterotoxins/therapeutic use , Escherichia coli Proteins/therapeutic use , Synapsins/therapeutic use , Animals , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , CHO Cells/drug effects , CHO Cells/metabolism , Cattle , Cricetinae , Drug Evaluation, Preclinical , Endocytosis , Enterotoxins/chemistry , Enterotoxins/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Female , G(M1) Ganglioside/metabolism , Lymphocyte Activation/drug effects , Lymphokines/metabolism , Male , Myelin Basic Protein/immunology , Myelin Basic Protein/toxicity , Peptide Fragments/chemistry , Peptide Fragments/therapeutic use , Protein Denaturation , Protein Folding , Protein Structure, Tertiary , Random Allocation , Rats , Rats, Wistar , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/therapeutic use , Single-Blind Method , Structure-Activity Relationship , Synapsins/chemistry , Synapsins/genetics , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunologyABSTRACT
The B subunit of Escherichia coli heat-labile toxin (LTB) may function as an efficient carrier molecule for the delivery of genetically coupled antigens across the mucosal barrier. We constructed vectors for the expression of LTB and LTBSC proteins. LTBSC is a fusion protein that comprises the amino acid sequence from the C-domain of rat synapsin fused to the C-terminal end of LTB. Both constructions have a coding sequence for a 6His-tag fused in-frame. LTBSC was expressed in E. coli as inclusion bodies. The inclusion bodies were isolated and purified by Ni2+-chelating affinity chromatography under denaturing condition. Purified LTBSC was diluted in several refolding buffers to gain a soluble and biologically active protein. Refolded LTBSC assembled as an active oligomer which binds to the GM1 receptor in an enzyme-linked immunosorbent assay (ELISA). Soluble LTB in the E. coli lysate was also purified by Ni2+-chelating affinity chromatography and the assembled pentamer was able to bind with high affinity to GM1 in vitro. LTBSC and LTB were fed to rats and the ability to induce antigen-specific tolerance was tested. LTBSC inhibited the specific delayed-type hypersensitivity (DTH) response and induced decreased antigen-specific in vivo and in vitro cell proliferation more efficiently than LTB. Thus, the novel hybrid molecule LTBSC when orally delivered was able to elicit a systemic immune response. These results suggest that LTBSC could be suitable for exploring further therapeutic treatment of autoimmune inflammatory diseases involving antigens from central nervous system.
