Abstract
Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.
Highlights
Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) produced by Vibrio cholerae show 80% homology at the primary amino acid sequence level and have similar 3D structures (Mekalanos et al, 1983; Sixma et al, 1991; Spangler, 1992)
We previously showed that both mutant LTs were immunogenic following either IG or IN administration, and that antibody titers induced by IN immunization were slightly higher than those induced by IG immunization (Park et al, 1999)
The abilities of mutant LTs to act as mucosal adjuvants were assessed by IG immunization in mice
Summary
Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) produced by Vibrio cholerae show 80% homology at the primary amino acid sequence level and have similar 3D structures (Mekalanos et al, 1983; Sixma et al, 1991; Spangler, 1992). Enhancement of cAMP levels alters ion transport, inducing secretion of water and chloride ions into the small intestine, resulting in traveler’s diarrhea or cholera (Spangler, 1992) Both CT and LT are strong immunogens by oral and other mucosal routes, via which most antigens are unable to induce immune responses. Substitution of Ala to His or Glu at position 72 resulted in a protein with a toxicity indistinguishable from that of wild-type LT, whereas Ala to Arg substitution at the same position greatly reduced toxicity (Giuliani et al, 1998) These observations suggest that mutations at key positions in the active site may affect ribosyltransferase activity, and that the specific amino acid used to replace wild-type residues may affect binding affinities for NAD or substrates. We investigated the mucosal adjuvant properties of the mutant LTs, using H. pylori urease as a coadministered antigen
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