Mucosal immunogenicity and adjuvant activity of the recombinant A subunit of the Escherichia coli heat-labile enterotoxin

The Escherichia coti heat-labile enterotoxin (LT) is an exceptionally effec tive mucosal immunogen and mucosal immunoadjuvant towards coadministered an tigens. Although, in general, the molecular basis of these properties is po orly understood, both the toxic ADP-ribosylation activity of the LTA subuni t and the cellular toxin receptor, ganglioside, G(M1)-binding properties of the LTB-pentamer have been suggested to be involved. In recent studies we found that G(M1)-binding is not essential for the adjuvanticity of LT, sugg esting an important role for the LTA subunit in immune stimulation. We now describe the immunomodulatory properties of recombinant LTA molecules with or without ADP-ribosylation activity, LTA(His)(10) and LTA-E112K(His)(10), respectively. These molecules were expressed as fusion proteins with an N-t erminal His-tag to allow simple purification on nickel-chelate columns. The ir immunogenic and immunoadjuvant properties were assessed upon intranasal administration to mice, and antigen-specific serum immunoglobulin-isotype a nd -subtype responses and mucosal secretory immunoglobulin A (IgA) response s were monitored using enzyme-linked immunosorbent assay. With respect to i mmunogenicity, both LTA(His)(10) and LTA-E112K(His)(10) failed to induce an tibody responses. On the other hand, immunization with both LT and the non- toxic LT-E112K mutant not only induced brisk LTB-specific, but also LTA-spe cific serum and mucosal antibody responses. Therefore, we conclude that lin kage of LTA to the LTB; pentamer is essential for the induction of LTA-spec ific responses. With respect to adjuvanticity, both LTA(His)(10) and LTA-E1 12K(His)(10) were found to stimulate serum and mucosal antibody responses t owards coadministered influenza subunit antigen. Remarkably, responses obta ined with LTA(His)(10) were comparable in both magnitude and serum immunogl obulin isotype and subtype distributions to those observed after communizat ion with LT, LT-E112K, or recombinant LTB. We conclude that LTA, by itself can act as a potent adjuvant for intranasally administered antigens in a fa shion independent of ADP-ribosylation activity and association with the LTB pentamer.