Abstract
Vaccines against cholera and other enteric diseases would be a major benefit for developing countries where these diseases are often life threatening, especially among children. Enterotoxigenic Escherichia coli (ETEC) causes acute watery diarrhea by colonizing the small intestine and producing heat-stable and heat-labile enterotoxins (ST and LT) . The LT-B, a binding subunit of E. coli LT is a highly active oral immunogen. The aim of this study was to understand molecular and cellular mechanisms for the induction and regulation of LT-B-specific immune responses following oral immunization.
We initially assessed E, coli LT as mucosal immunogen and as adjuvant in mice. Oral administration of LT to BALB/c (H-2d) mice induced high mucosal IgA and serum IgG antibody responses. Analysis of LT-B-specific CD4+ T helper (Th) cells from Peyer's patches (PP) or spleen (SP) revealed a mixed Thl (IFN-γ) and Th2 (IL-4 and IL-5) cell pattern.
The B cell and Tcell epitopes of LT-B which induce effective secretory IgA (S-IgA) responses were examined in the next experiment. LT-B-specific S-IgA of BALB/c mice orally immunized with recombinant LT-B expressed in S. typhimurium showed major recognition of peptide starting at residue 36. PP CD4+ T cells cultured with the peptide covering residues 34 to 43 of the LT-B molecule produced large amounts of IL-5 and IFN-γ. These findings indicated that the residues 34 to 43 of the LT-B molecule enhanced the production of both Thl-and Th2-type cytokines. According to these findings, a peptide [peptide (26-45) ] encompassing both the B-cell and T-cell epitopes was synthesized as a possible candidate for oral vaccine. When BALB/c (I-Ad) mice as well as three strains of B10 congenic mice [B10 (I-Ab), B10. D2 (I-Ad), and B10. BR (I-Ak) ] were orally immunized with the peptide, serum IgG and S-IgA responses were elicited not only to the peptide itself, but also to the native LT. In addition, PP CD4+ T cells from all the stains of B10 congenic mice as well as BALB/c mice induced strong peptide-specific T cell proliferation and cytokine synthesis, suggesting the promiscuous binding of the peptide to MHC class II molecule.
A possibility for induction of oral tolerance by mucosal administration with the LT-B peptide was examined by a multiple low dose feeding of the immunogenic peptide (26-45) . Following the multiple oral feeding with the LT-B peptide induced systemic unresponsiveness in BALB/c mice resulting in diminished serum IgG responses and maintenance of appropriate mucosal IgA responses. CD4+ T cells from SP of the tolerized mice failed to proliferate, whereas PP CD4+ T cells responded to the peptide. RT-PCR revealed that the PP CD4+ T cells expressed significant levels of mRNA for IFN-γ, IL-2, IL-4, and TGF-β. These results indicated that SP CD4+ T cells induced a state of systemic T cell anergy.
