Folding and Immunogenicity of Zinc-Finger Peptide Constructs Corresponding to Loop Regions of the Protein Antigens LDH-C\u3csub\u3e4\u3c/sub\u3e and β-hCG

This paper describes our continuing studies on stabilization of peptide structures in supersecondary conformations that are designed to mimic conformational antigenic epitopes. In this work we have used the consensus Cys2His2 zinc-finger peptide motif as a template to engineer and synthesize antigenic loop peptide segments from two protein antigens, lactate dehydrogenase C4 isozyme (LDH-C4) and human chorionic gonadotropin β subunit (β-hCG). Confirmation that the engineered peptide constructs assumed a zinc-finger conformation was obtained by absorption spectroscopy of the Co2+ complexes. The circular dichroism (CD) spectra of the free peptides show random coil conformations, while the Zn2+-complexed peptides acquired the zinc-finger motif upon titration with Zn2+, as evidenced by the appearance of absorbances indicating α-helix and some β-conformation. No peptide aggregation was observed, as these peptides were monomeric under all conditions tested. In order to examine the immunogenicity of the zinc-finger constructs, one sequence from LDH-C4 (ZFLMVF) and two sequences from β-hCG (ZF2TT3 and ZF4TT3) were selected and chimeras were synthesized to incorporate promiscuous T-cell epitopes from either tetanus toxoid or measles virus. The ZFLMVF construct was highly immunogenic in rabbits, and the ZF2TT3 and ZF4TT3 peptides were highly immunogenic in both mice and rabbits, eliciting high-titer antipeptide antibodies specific for their immunogenic sequences. However, the antibodies raised to the zinc-finger constructs showed minimal reactivity against their respective native protein antigens as determined by ELISA. This is surprising in the case of β-hCG, since the ZF2 zinc-finger peptide was an effective inhibitor of binding of anti-β-hCG-loop(38-57) antibodies to whole hCG, as assessed by a competitive inhibition radioimmunoassay. This implies that, although the cyclized 40-52 sequence from βhCG and the zinc-finger peptide ZF2 exhibit similar conformations in solution, the zinc-finger engineered loop is apparently not in a sufficiently correct conformation for antibody recognition of native hCG. Our results with the LDH-C4 zinc finger loop imply that antibody recognition of antigen involves specific side-chain interactions that must be maintained by a precise conformation