Most of the potent neutralizing mAbs were generated by immunization of IFN-/ R -/- C57BL/6 mice with DENV twice and booster with recombinant domain name III ,  , whereas most of the less potent neutralizing mAbs were generated by immunization of WT BALB/c mice , , , C. Predominant Epitopes in Polyclonal Human Sera To further explore the possibility that this assay can be employed to investigate the predominant epitope of anti-E Abs in polyclonal human sera, we examined serum from a confirmed DENV1 secondary contamination case. and Western blot analyses.(TIF) pntd.0001447.s003.tif (1.9M) GUID:?C50301D5-6ABE-46DF-988A-95189450DEE1 Physique S4: Binding specificity and predominant TNC epitope recognized by anti-E Abs in human sera from dengue cases. Shown are cases of DENV2 (A,B,C), and DENV3 (D,E,F). (A,D) Binding specificity was examined by Western blot analysis as explained in Methods. Lysates of 293T cells transfected with pCB-D1 (D1 tr) were also included. (B,E) Dot blot assay offered as in Fig. 1A and 1C to 1E (except that WT dot in row 8C and 153NA dot in row 2H were omitted) was probed with the tested serum or mixed sera, which consisted of a pool of 9 sera from confirmed dengue patients . The relative intensities of two-fold dilutions of WT dots in row 1 were presented as in Fig. 1D. R.I. of each mutant was shown as in Fig. 1E. One representative experiment of two was shown. (C,F) Capture ELISA using WT or mutant VLPs was offered as in Fig. 1F. Upper graph in panel C shows comparable amounts of WT and mutant VLPs added.(TIF) pntd.0001447.s004.tif (2.3M) GUID:?0448EFBC-8E92-4004-9FF6-4368FFF04C94 Table S1: Comparison of Etofenamate epitopes, neutralization potency and immunization protocol of CR/sCR and TS mAbs recognizing domain name III of DENV E protein. (DOC) pntd.0001447.s005.doc (89K) GUID:?B2A1D243-72FE-4A80-AAA2-33EEAA1BA396 Abstract Background The envelope (E) protein of dengue computer virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain name III or domain name I/II alone have reported several epitopes of monoclonal Etofenamate antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. Methodology/Principal Findings We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three acknowledged a novel epitope including residues (Q211, D215, P217) at the central interface of domain name II, and three acknowledged residues at both domain name Etofenamate III and the lateral ridge of domain name II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol acknowledged multiple residues in A strand or residues in C strand/CC loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Conclusions/Significance Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level. Author Summary Dengue computer virus is the leading cause of arboviral diseases worldwide. The envelope protein is the major target of neutralizing antibodies and vaccine development. While previous studies have reported several epitopes on envelope protein, the possibility of interdomain epitopes and the relationship of epitopes to neutralizing potency remain unexplored. We developed a high throughput dot blot assay by using 67 alanine mutants of surface-exposed envelope residues as a systematic approach to identify epitopes recognized by mouse monoclonal antibodies and polyclonal human sera. Our results suggested the presence of interdomain epitopes more frequent than previously appreciated. Compared with monoclonal antibodies generated by traditional protocol, the potent neutralizing monoclonal antibodies generated by a new protocol showed several unique features of their epitopes. Moreover, the predominant epitopes of antibodies against envelope protein in polyclonal sera can be recognized by this assay. These findings have implications for future development of epitope-specific diagnostics and epitope-based dengue vaccine, and add to our understanding of humoral immune responses to dengue computer virus at the epitope level. Introduction Dengue computer virus (DENV) belongs to the genus in the family em Flaviviridae Etofenamate /em . The four DENV serotypes (DENV1, DENV2, DENV3, and DENV4) are the leading.