TANDEM REPEATS OF T-HELPER EPITOPES ENHANCE IMMUNOGENICITY OF FUSION PROTEINS BY PROMOTING PROCESSING AND PRESENTATION

Empirical findings have shown that recombinant chimeric proteins may b e made more immunogenic if T helper epitopes are incorporated as tande m repeats. In the present study we investigated the mechanisms respons ible for the enhanced immunogenicity of fusion proteins composed of th e heat-stable enterotoxin of enterotoxigenic E. coli (STa) linked to m ultiple copies of the ovalbumin(323-339) T helper epitope (oval and a connecting dimer of an Ig-binding region of Staphylococcus nta eus pro tein A (ZZ), which were previously shown to stimulate strong anti-STa titres in mice. We used B cell and macrophage cell lines as APC and IL -2 production by ova-specific T cells as our read-out system. Fusion p roteins containing four repeated T helper epitopes were found to be th e most immunogenic and resulted in 50-fold higher IL-2 production than constructs with a single T helper epitope. Under limiting APC conditi ons the construct with four epitopes was the best inducer of IL-2, ind icating that this construct was most effectively processed by the APC. Analysis of IL-2R alpha expression by flow cytometry confirmed that f our copies gave the highest frequency of activated T cells in culture, indicating a direct correlation between ability to activate T cells a nd IL-2 production in culture. Also in vivo, the fusion protein with f our epitopes exhibited the strongest T cell priming effect. Moreover, both in vitro and in vivo, the ZZ construct was found to serve as an e fficient means for targeting of the fusion proteins to B cells, thereb y allowing access to the Ig receptor uptake pathway for Ag. The presen t study provides direct evidence that fusion proteins can be construct ed to optimize processing in the individual APC and enhance activation of clonal T cells. (C) 1997 Published by Elsevier Science Ltd.