Mapping of peptide sequences using the Peptiope server against the 3D crystal structure of the prM-E heterodimer at neutral pH (PDB 3C6E) identified three main clusters of predicted epitope location. Since Pepitope assumes all input peptides mimic surface residues; all buried residues are eliminated from the search. The Figure 4 . Localization of D29 Fab-IgG predicted epitopes in 3D crystal structures of DENV2 prM-E heterodimer (PDB 3C6E) and the binding specificity of peptide-phages. (A) Clusters of conformational epitopes predicted by Pepitope server are displayed on the prM-E heterodimer crystal structure at neutral pH with their solvent-accessible surfaces highlighted. The prM is pink, EDI is red, EDII is yellow, EDIII is blue, FP is cyan. (B) The binding specificity of peptide-phages (P1-P9) to D29 Fab-IgG was tested in a direct ELISA format with 10 mg/ml of D29 Fab-IgG, h3H5, m2H2 and non-DENV specific human antibody (Hu) immobilized on a Maxisorb plate. Ability of peptide-phages to inhibit D29 Fab-IgG binding to DENV2 was investigated by (C) ELISA and (D) Western blot analysis. For ELISA, peptide-phages (10 12 pfu/ml) were incubated with D29 Fab-IgG for 1 hr at RT before application to immobilized DENV2 for 5 min at RT. Bound D29 Fab-IgG was detected with HRP-conjugated anti-Human IgG-Fc. The percentage of inhibition of D29 Fab-IgG binding by the peptide-phages shown is the average of three experiments. Error bars represent the standard errors of the mean (***p-value ,0.005). For Western blot analysis, 0.5 mg/ml of D29 Fab-IgG was incubated with 8610 6 pfu of purified DENV2, 5% SM or 4610 11 pfu of peptide-phage clones for 1 hr at RT before applying to membrane transblotted with DENV2 viral lysate for 30 min at RT. doi:10.1371/journal.pone.0033451.g004 peptides with the KXPXW motif could mostly be mapped to the highest scoring Cluster 1 located at the prM/EDII interface. A few exceptions mapped to Cluster 3, most probably due to poorer sequence match and a lower alignment score. The remaining two motifs were mapped to the second highest scoring Cluster 2 that spans EDI and EDII (Fig. 4A) .