Identification of peptides mimicking the antigenicity and immunogenicity of conformational epitopes on Japanese encephalitis virus protein using synthetic peptide libraries

Monoclonal antibodies (mAbs) N.03 and N.08 that recognize conformational epitopes on the prM protein of Japanese encephalitis virus (JEV) were analyzed to identify their peptide ligands by using a novel approach that combined two different synthetic peptide libraries. Immunoscreening of a library containing 20(5) sequences of pentapeptides revealed that the ligands for N.03 and N.08 had motif sequences, (Y/W/F)GG(I/L/M) and (N/Q)WY(D/E), respectively. To select higher-affinity ligands, we synthesized and screened another type of library with 20 peptide mixtures that were based on the identified motif, where only one amino acid position was defined; and the process was reiterated for the remaining undefined positions. Consequently, the peptides YGGIYMNG and QWYDDR were identified as peptide ligands of N.03 and N.08, respectively. These peptides bound specifically to the antigen-combining sites of the mAbs as confirmed by competitive binding assays. Mouse antisera directed against the peptide YGGIYMNG specifically recognized JEV, while those against QWYDDR did not. These data demonstrated that peptide ligands which reproduce or mimic the immunogenicity as well as the antigenicity of conformational epitopes can be at least partly identified using this approach. This approach may be useful for analyzing conformational epitopes, which are generally difficult to characterize, and might provide a step toward vaccine development when applied to protective mAbs.