Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library.

Shiga toxins (Stxs) are involved in the pathogenesis of hemolytic-uremic syndrome and other severe systemic complications following enterohemorrhagic Escherichia coli infection in humans. Passive immunotherapies using monoclonal antibodies have been shown to be effective for neutralizing the toxic effects of Stxs. However, animal-derived monoclonal antibodies are sometimes immunogenic and their production is both laborious and expensive. We here report the isolation of single-chain variable fragment antibodies against Stxs by screening a phage display library constructed from a naïve human repertoire. An antibody among the selected clones designated B22 bound to the binding subunits of both Stx-1 and Stx-2, and strongly neutralized the cytotoxicity of Stx-1. This is the first example of a monovalent antibody showing Stx-neutralizing activity. The B22 antibody is also completely naturally occurring in human, which reduces the possibility of adverse immunological effects, and can be easily produced using bacterial protein synthesis systems.

Mutant Escherichia coli enterotoxin as a mucosal adjuvant induces specific Th1 responses of CD4+ and CD8+ T cells to nasal killed-bacillus calmette-guerin in mice.

On single nasal immunization of mice with killed-bacillus calmette-guerin (BCG) plus a mutant Escherichia coli enterotoxin, delayed-type hypersensitivity was induced and BCG-infection decreased. Spleen cells, particularly CD4+ T cells among them produced IL-2, IFNgamma and TNFalpha in response to the killed-BCG or purified protein derivatives. CD8+ T cells including cytotoxic T lymphocytes produced IFNgamma and TNFalpha. However, both types of T cells reacted a little to Ag85B. The mutant induces cellular immunity to nasal killed-BCG vaccine and decreases BCG-infection. CD4+ and CD8+ T cells produce cytokines effective for tuberculosis. Although killed-BCG loses some antigens like Ag85B, nasal killed-BCG plus the mutant is useful for tuberculosis.

Induction of thymus-derived gammadelta T Cells by Escherichia coli enterotoxin b subunit in peritoneal cavities of mice.

We examined the activation of intraperitoneal T cells in BALB/c mice by the Escherichia coli enterotoxin B subunit, which induced a specific Th2 type of T-cell response to intraperitoneally coadministered bovine immunoglobulin G. The numbers of both gammadelta and alphabeta T cells increased significantly after intraperitoneal administration of the B subunit in a time-dependent manner; these numbers were not affected by the B-subunit G33D mutant, which is defective in GM1 ganglioside-binding ability. Early after administration a small number of gammadelta T cells produced either interleukin-4 (IL-4) or gamma interferon, while late after administration primarily IL-10-producing gammadelta T cells were detected. gammadelta T cells induced by the B subunit did not express a characteristic V gene over the time course of the study. The induction of gammadelta T cells did not occur in athymic nu/nu mice but could be induced upon transplantation of fetal AKR thymus-like alphabeta T cells. gammadelta T cells in athymic nu/nu mice with a fetal thymic graft predominantly expressed the donor Thy-1.1 antigen but not the host Thy-1.2 antigen. The induction of these T cells, however, could not be restored by coadministration of the B subunit with peritoneal cells from normal mice. These results suggest that the B subunit activates intraperitoneal gammadelta and alphabeta T cells in a manner dependent upon its ability to bind to GM1 ganglioside. gammadelta T cells induced by the B subunit are Th2-type cells derived from the thymus. These gammadelta T cells may be functionally involved in specific Th2 responses to the B subunit, which possibly acts as an adjuvant through the influence of alphabeta T cells.