Underlining denotes a LB5010 as described (4) and then introduced into rationally attenuated BRD915 (BRD915 was incubated at 37C with aeration in LB containing ampicillin until early/mid-log phase (optical density at 600 nm, 0

Underlining denotes a LB5010 as described (4) and then introduced into rationally attenuated BRD915 (BRD915 was incubated at 37C with aeration in LB containing ampicillin until early/mid-log phase (optical density at 600 nm, 0.3 to 0.5). various cell lines in vitro (46). A striking feature of these toxins is the repetitive nature of the amino acid sequence at the carboxyl terminus of the protein (1, 13). In the case of toxin A, this region is composed of 38 contiguous repeat sequences which encode the receptor-binding Vegfb domain of toxin A (33, 40). One of these repeat sequences, the class IIB repeat, is of particular interest because a synthetic decapeptide encoding amino acids conserved within this repeat was shown to promote cellular attachment in vitro (53). Toxin A has been shown to be the primary mediator of tissue damage within the gastrointestinal tract, as direct administration of toxin A alone induces tissue damage characteristic of infection (35, 37). Recently, the direct binding of toxin A to human colonic epithelial cells has been demonstrated (42). To date, the experimental vaccine strategies employed to induce a protective anti-toxin A response have been limited, although parenteral immunization with small amounts of purified toxin A has been shown to solidly protect rabbits against toxin-induced death (26). However, this form of immunization was unable to EGFR Inhibitor prevent toxin-mediated mucosal damage. Indeed, mucosal damage appeared to be a prerequisite for protection, allowing toxin-neutralizing antibodies to be released from serum and into the intestinal lumen. This result suggests that the induction of a toxin-neutralizing, secretory immunoglobulin A (IgA)-mediated response at the mucosal surface, to prevent tissue damage, would be desirable. Toxin A-specific IgA harvested from human mucosa has been shown to inhibit toxin A from binding to intestinal brush border (25), thus validating the principle of anti-toxin A mucosal immunity. Mucosal immunization with toxoid vaccines has also been shown to protect against mucosal challenge by whole organisms (18, 45). However, chemically detoxified immunogens are not wholly satisfactory due to possible residual toxicity and the random structural and chemical modifications which occur to the antigen. In addition, formalin-inactivated molecules that cannot bind to or target mucosal surfaces have been described as being generally poorer mucosal immunogens than molecules that can successfully target receptors on the mucosal surface (8). The nontoxic C-terminal repeat region of toxin A has been reported to be a good vaccine EGFR Inhibitor candidate. Immunization with a recombinant protein EGFR Inhibitor expressing 33 of the 38 C-terminal repeats generated a partially protective anti-toxin A response (33). Also, a synthetic peptide containing 10 conserved amino acids from the class IIB repeat stimulated toxin-neutralizing antibodies (53). Several studies have shown the induction of a toxin-neutralizing response to protect against whole-organism challenge in vivo (18, 45). Our goal, therefore, was to induce an antibody response against nontoxic fragments of the toxin A repeat region which would be able to neutralize the effects of the whole molecule systemically and at the mucosal surface. Such a fragment would be desirable as a component of a recombinant vaccine. We have previously shown all 14 C-terminal repeats of toxin A (14CDTA) to be immunogenic when fused genetically to the nontoxic C-terminal domain (TETC) from tetanus toxin (TT) and delivered to the mucosal surface by attenuated (48). In the present study, we evaluate the immunogenicity of 14CDTA when administered directly to the murine nasal mucosa in a purified form. It is well documented that other bacterial toxins which bind to mucosal surfaces, such as heat labile toxin (LT) from LB5010 (BL21 (DE3) was obtained from Novagen, and plasmid pRSET-A was supplied by.