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SUMMARYEscherichia coli (E. coli ) heat-labile toxin (LT ) is a potent mucosal immunogen and immunoadjuvant towards co-administered antigens. LT is composed of one copy of the A subunit, which has ADP-ribosylation activity, and a homopentamer of B subunits, which has affinity for the toxin receptor, the ganglioside G M1 . Both the ADP-ribosylation activity of LTA and G M1 binding of LTB have been proposed to be involved in immune stimulation. We investigated the roles of these activities in the immunogenicity of recombinant LT or LTB upon intranasal immunization of mice using LT/LTB mutants, lacking either ADP-ribosylation activity, G M1 -binding affinity, or both. Likewise, the adjuvant properties of these LT/LTB variants towards influenza virus subunit antigen were investigated. With respect to the immunogenicity of LT and LTB, we found that G M1 -binding activity is essential for effective induction of anti-LTB antibodies. On the other hand, an LT mutant lacking ADP-ribosylation activity retained the immunogenic properties of the native toxin, indicating that ADP ribosylation is not critically involved. Whereas adjuvanticity of LTB was found to be directly related to G M1 -binding activity, adjuvanticity of LT was found to be independent of G M1 -binding affinity. Moreover, a mutant lacking both G M1 -binding and ADPribosylation activity, also retained adjuvanticity. These results demonstrate that neither ADPribosylation activity nor G M1 binding are essential for adjuvanticity of LT, and suggest an ADP-ribosylation-independent adjuvant effect of the A subunit. INTRODUCTIONinvestigators have shown that the enzymatic activity of LTA does not play a major role in the immunogenicity of LT.8-11 The Escherichia coli (E. coli ) heat-labile toxin (LT ) and Vibrio On the other hand, recombinant LTB alone is clearly less cholerae cholera toxin (CT ) are exceptionally potent mucosal immunogenic than LT mutants which lack ADP-ribosylation immunogens and immunoadjuvants.1-5 In addition, LT and activity,12 suggesting that the presence of LTA, but not CT have been found to abrogate tolerance towards necessarily its enzymatic activity, does contribute to the co-administered antigens.1,3 Both are heterohexameric proteins antitoxin antibody response. The role of the A subunit in composed of one copy of the A subunit, which has ADPadjuvanticity is even less clear. Several studies have shown ribosylation activity, and five copies of the B subunit. The that recombinant LTB and CTB lack the capacity to stimulate B-pentamer has high affinity for the toxin receptor, ganglioside systemic and mucosal antibody responses towards G M1 . Delivery of the A subunit to the cytosol of the target cell co-administered antigens.13-18 Furthermore, using an LT results in persistent synthesis of cAMP.6,7 mutant lacking ADP-ribosylation activity, Lycke et al.16 Even though the role of the individual subunits of LT showed that the adjuvant effect of LT and CT is directly and CT in the toxic mechanism are well defined, their role in linked to the enz...

SUMMARYThe Escherichia coli heat-labile enterotoxin (LT ) is an exceptionally effective mucosal immunogen and mucosal immunoadjuvant towards coadministered antigens. Although, in general, the molecular basis of these properties is poorly understood, both the toxic ADP-ribosylation activity of the LTA subunit 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, suggesting 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-terminal His-tag to allow simple purification on nickel-chelate columns. Their immunogenic and immunoadjuvant properties were assessed upon intranasal administration to mice, and antigen-specific serum immunoglobulinisotype and -subtype responses and mucosal secretory immunoglobulin A (IgA) responses were monitored using enzyme-linked immunosorbent assay. With respect to immunogenicity, both LTA(His) 10 and LTA-E112K(His) 10 failed to induce antibody responses. On the other hand, immunization with both LT and the non-toxic LT-E112K mutant not only induced brisk LTBspecific, but also LTA-specific serum and mucosal antibody responses. Therefore, we conclude that linkage of LTA to the LTB pentamer is essential for the induction of LTA-specific responses. With respect to adjuvanticity, both LTA(His) 10 and LTA-E112K(His) 10 were found to stimulate serum and mucosal antibody responses towards coadministered influenza subunit antigen. Remarkably, responses obtained with LTA(His) 10 were comparable in both magnitude and serum immunoglobulin isotype and subtype distributions to those observed after coimmunization 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 fashion independent of ADP-ribosylation activity and association with the LTB pentamer. INTRODUCTIONB subunits. The A subunit carries the toxic ADP-ribosylation activity of the toxin, while the B-pentamer has high affinity for The Escherichia coli heat-labile enterotoxin (LT ) and its close the cellular toxin receptor, ganglioside G M1 . Intoxication of homologue from Vibrio cholerae, cholera toxin (CT ), are wellintestinal epithelial cells with LT or CT results in persistent known powerful mucosal immunogens and adjuvants. Both synthesis of cAMP and concomitant excessive electrolyte and toxins have proven to be valuable tools for unravelling mechanfluid secretion to the intestinal lumen (for reviews see refs. 4,5). isms of induction of mucosal immune responses, and have Over the last 10 years, several strategies have been develbecome standard adjuvants for experimental mucosal vacoped in order to investigate whether the toxic properties of cines.1-3 How...

The Escherichia coli heat-labile enterotoxin (LT) is a potent inducer of mucosal immune responses. In a previous study (L. DeHaan, W. R. Verweij, M. Holtrop, E. Agsteribbe, and J. Wilschut, Vaccine 14:620-626, 1996), we have shown that efficient induction of an LTB-specific mucosal immune response by LT requires the presence of the LTA chain, suggesting a possible role of the enzymatic activity of LTA in the induction of these responses. In the present study, we generated LT mutants with altered ADP-ribosylation activities and evaluated their immunogenicity upon intranasal administration to mice. The results demonstrate that the mucosal immunogenicity of LT is not dependent on its ADP-ribosylation activity.