Abstract
Japanese encephalitis virus (JEV) is a mosquito-borne neurotropic flavivirus. The envelope (E) protein is the major surface protein that dominantly induces neutralizing antibody responses against the JEV infection. The lateral surface of domain III of the E protein was presumed to be responsible for conformational neutralizing epitopes and receptor-binding sites. Identification of conformational epitopes on domain III is important for development of new JEV vaccines, but still has practical difficulties. In the present study, the approach to identify mimotope and site-directed mutagenesis of domain III-Fab complexes were performed to investigate the structural characteristics of neutralizing epitopes on the JEV domain III. The modeled structures of six mimotope peptides that selected from the phage display peptide libraries with a neutralizing monoclonal antibody (mAb) E3.3 mimic a conformational region at the lateral surface of the JEV domain III. Alanine scanning mutagenesis of the mAb E3.3-reactive peptide ligands demonstrated that a novel cis-proline turn structure constrained the conformation of selected peptide ligands to induce JEV-specific neutralizing antibodies in mice. Furthermore, site-directed mutagenesis of recombinant domain III indicated that Ser331 and the negative charged side-chain of Asp 332 were the key residues constituted the functional epitope determinants interacting with mAb E3.3. Bacterial expression of recombinant E3.3 Fab also confirmed the molecular interactions of the ArgH94 in CDRH3 of mAb E3.3 with Ser331 and Asp332 on domain III. Together with the results of mimotope peptides and mutational analysis, the similar structures between conformational epitopes for neutralizing mAbs E3.3 and 2H2 are identified in four segments (Loops AxA, BxC, DxE and FG) at the lateral surface of domain III. The results of the present study for molecular determination of neutralizing epitopes in the JEV domain III will be useful for rationally developing new JEV vaccines.