GENETICALLY-ENGINEERED NONTOXIC VACCINE ADJUVANT THAT COMBINES B-CELLTARGETING WITH IMMUNOMODULATION BY CHOLERA-TOXIN A1 SUBUNIT

Cholera toxin (CT) is an exceptionally potent adjuvant but, unfortunat ely, also very toxic. Here we present a powerful new approach to separ ate toxicity from adjuvanticity by constructing a fusion protein that combines the enzymatically active cholera toxin A1 subunit (CTA1) with targeting to B cells. The CTA1 was genetically linked at its C-termin al end to two Ig-binding domains, DD, of staphylococcal protein A and produced in Escherichia coli. The highly purified, monomeric CTA1-DD f usion protein, with a molecular mass of 37 kDa, was found to exhibit s trong ADP-ribosyltransferase activity and bound, via the DD moiety, to both Fc and Fab fragments and to all IgG subclasses-IgE, IgA, and IgM . After i.v. injection of the fusion protein, FACS analysis revealed b inding of CTA1-DD to splenic IgM(+) B cells, but not CD3(+) T cells, i ndicating cell-specific targeting in vivo. Strikingly, we found that t he adjuvant ability of CTA1-DD to enhance systemic IgG as well as muco sal IgA responses to the unrelated Ags, OVA, or keyhole limpet hemocya nin, administered i.v or intranasally, was comparable to that of intac t CT. In addition, the enhancing effect on specific IgG1, IgG2a, and I gG2b responses mimicked that of CT and suggested involvement of both T h1 and Th2 CD4(+) T cell activity. The CTA1-DD, as well as CT, up-regu lated expression of the CD80 and CD86 molecules on the targeted B cell s, indicating that enhanced T cell costimulation may be responsible fo r the adjuvant effect. Contrary to CT, however, CTA1-DD was completely nontoxic. Thus, the CTA1-DD adjuvant should find general applicabilit y in systemic and mucosal vaccines, and the strategy used may also be explored for other regimens requiring targeted immunomodulation.