CTA1-DD; A novel vaccine adjuvant system based on ADP-ribosyltransferase activity and B cell targeting
Sammanfattning: The ADP-ribosylating enterotoxins, cholera toxin (CT) and E. coli heat-labile toxin (LT) are among the most powerful immunogens and adjuvants yet described. An innate problem, though, 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. The mechanisms for the adjuvant function of the ADP-ribosylating enterotoxins, cholera toxin (CT) and E. coli heat-labile toxin (LT) are still incompletely understood. Whereas some investigators have separated adjuvanticity from toxicity by disrupting the enzymatic activity of the A1-subunit by site-directed mutagenesis, we have constructed and evaluated the adjuvant and toxic functions of a molecule that combines the full enzymatic activity of the cholera toxin A1-subunit with a B cell targeting moiety in a gene fusion protein, the CTA1-DD adjuvant. We found that CT can be made non-toxic by targeting the ADP-ribosyltransferase activity of CT to Ig-bearing B cells in vivo. The novel fusion protein CTA1-DD hosted systemic and mucosal adjuvant functions comparable to those of intact CT, but, in contrast to CT, CTA1-DD was completely non-toxic. We also found that enzymatically inactive mutants of the CTA1-DD fusion protein had no adjuvant function in vivo, directly supporting the idea that the ADP-ribosyltransferase activity is important for adjuvanticity. Furthermore, our fusion protein lost its adjuvant function when mutations were introduced which impaired the binding of CTA1-DD to immunoglobulin. Although CTA1 in CT is known to ADP-ribosylate the GTP-binding protein Gsa, leading to an activation of adenylate cyclase and subsequent increase of intracellular cAMP, we failed to show an effect on cAMP levels with CTA1-DD. The CTA1-DD fusion protein did not appear to form immune-complexes or bind to soluble Ig following immunization, but rather it bound directly to B cells. In normal mice, both CT and CTA1-DD, but not the enzymatically inactive CTA1-R7K-DD mutant, were efficient enhancers of TD as well as TI- responses and both promoted germinal center formation following immunizations. In contrast to CT, CTA1-DD exerted strong immunoenhancing effects in athymic mice and prevented apoptosis in antigen-receptor activated B cells by upregulating the expression of bcl-2. CTA1-DD also induced tyrosine phosphorylation of a specific substrate/s and released intracellular Ca2+-stores after binding to B cells. Furthermore, CTA1-DD ADP-ribosylated the Gsa protein, but also other G proteins in the membrane and cytosol. These results demonstrate that adjuvanticity and toxicity of CT can be separated. By excluding the B-subunit and via direct targeting of the enzymatic activity of the CTA1-subunit to B cells, we have achieved full adjuvanticity with no toxicity. The adjuvant effect of CTA1-DD is dependent on specific targeting to B cells and ADP-ribosyltransferase activity. Whether it is the anti-apoptotic or the enhanced CD4+ T cell priming effect of CTA1-DD that is responsible for the adjuvant effect remains to be investigated. We believe that CTA1-DD represents an important advancement in immunomodulation and vaccine adjuvant research.
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