Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

Abstract

BackgroundThe envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored.Methodology/Principal FindingsWe 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 recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain 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 recognized 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/SignificanceOur 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.

Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection

“…ZIKV-immune plasma was also found to cross-react with ZIKV and DENV1-4 E proteins ( fig. S11), possibly because of the abundant EDI/II cross-reactive antibody response (21,(26)(27)(28).…”

Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination

“…We then probed the folding state of the different proteins using mAb 4G2, which recognizes a DII conformational epitope on the fusion loop of all serotypes (Henchal et al, 1985;Lin et al, 2012). 4G2 recognized secreted DI/ DII and sE proteins from DENV3 and DENV4, as well as the secreted chimeras 2DI/3DII and 2sE(3DII) (Fig.…”

MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus

“…Asp215 is important for infectivity 42 , a proven 41 and predicted 47 epitope, and a membranotropic region of the E protein (peptide 29) 67 .…”

“…Characterization of α -helices in E-proteins using techniques (PAGAL 44, 45 ) previously applied to the Ebola virus 46 , revealed that α 1 in ZIKV-E and DENV-E proteins is not conserved in the sequence space. Furthermore, α 1 is perturbed in the post-fusion protein in DENV2-E protein 12 , and includes a known epitope that is not part of the pre-fusion α 1 41, 42, 47 . PAGAL analysis also highlights a cationic β -sheet within a putative GAG-binding domain 48, 49 , which consists of a pair of arginine residues that have significant electrostatic polarity reversal 48, 49 .…”