Adjuvanticity of the cholera toxin A1-based gene fusion protein, CTA1-DD, is critically dependent on the ADP-ribosyltransferase and Ig-binding activity.
The ADP-ribosylating enterotoxins, cholera toxin (CT) and Escherichia coli heat-labile toxin, are among the most powerful immunogens and adjuvants yet described. An innate problem, however, is their strong toxic effects, largely due to their promiscuous binding to all nucleated cells via their B subunits. Notwithstanding this, their exceptional immunomodulating ability is attracting increasing attention for use in systemic and mucosal vaccines. Whereas others have separated adjuvanticity from toxicity by disrupting the enzymatic activity of the A1 subunit by site-directed mutagenesis, we have constructed a nontoxic molecule that combines the full enzymatic activity of the A1 subunit with a B cell targeting moiety in a gene fusion protein, the CTA1-DD adjuvant. Despite its more selective binding properties, we found comparable adjuvant effects of the novel CTA1-DD adjuvant to that of CT. Here we unequivocally demonstrate, using a panel of mutant CTA1-DD molecules, that the immunomodulating ability of CTA1-DD is dependent on both an intact enzymatic activity and the Ig-binding ability of the DD dimer. Both agents, CT and CTA1-DD, ADP-ribosylate intact B cells. However, contrary to CT, no increase in intracellular cyclic AMP in the targeted cells was detected, suggesting that cyclic AMP may not be important for adjuvanticity. Most remarkably, CTA1-DD achieves similar immunomodulating effects to CT using a ganglioside-GM1 receptor-independent pathway for internalization.
Agren
L C
LC
Department of Medical Microbiology and Immunology, University of Göteborg, Sweden.
Ekman
L
L
Löwenadler
B
B
Nedrud
J G
JG
Lycke
N Y
NY
D000276:Adjuvants, Immunologic / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology / Q000276:immunology; D001666:Binding Sites, Antibody / Q000235:genetics; D002772:Cholera Toxin / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D007136:Immunoglobulins / Q000378:metabolism; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000378:metabolism* / Q000494:pharmacology