Fusion Proteins with Heterologous T Helper Epitopes. Recombinant E. coli Heat-Stable Enterotoxin Proteins

Abstract

Fusion proteins containing specific B cell and T cell epitopes were used to examine how the intramolecular arrangement of T and B cell epitopes within a chimeric protein influences antigen-specific B cell antibody responses as well as specific T cell activation. Chimeric proteins, containing single or multiple copies of the T_h epitope ovalbumin 323-339 (ova) linked at different positions to STa, the heat-stable enterotoxin of E. coli, were compared with respect to their ability to induce STa-specific antibody production and to induce ova-specific T cell activation.

Chimeric proteins induced ova-dependent antibody production against STa at the amino terminal end, irrespective of the positioning of ova. Multiple tandem copies of ova in any position led to increased levels of antibody production against this epitope. In contrast, T cell help for antibody production against a second B cell epitope at the carboxy terminus of the fusion proteins was more effective after insertion of multiple copies of ova in a distal than in an adjacent position. A fusion protein, containing four copies of ova effectively elicited T cell help for antibody production against both examined B cell determinants, showing that activated T_h cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein.

T cell recognition of ova in all chimeric peptides, independently of its position, followed the same pattern of genetic restriction (i.e. immunodominant in H-2^d and nonimmunogenic in H-2^k) as in the native ovalbumin molecule. At high doses, the level of ova-specific T cell activation in LNC of immunized mice was similar, irrespective of the number of ova copies in the fusion proteins. In contrast, at low antigen doses chimeric proteins containing multiple copies of ova effectively primed ova-specific T cells at 20-fold lower concentrations than did fusion proteins containing a single copy of ova. Furthermore, when the ova-containing constructs were presented to a ova-specific T cell line by A20 B lymphoma cells, an increased level of IL-2 production by the clonal T cells in response to multiple copies of ova in the chimeric antigens was observed.

Key Words:

• recombinant fusion protein
• T cell epitopes
• B cell epitopes
• immunogenicity
• ova
• STa