Comprehensive mapping of functional epitopes on dengue virus glycoprotein E DIII for binding to broadly neutralizing antibodies 4E11 and 4E5A by phage display

Abstract

Here we investigated the binding of Dengue virus envelope glycoprotein domain III (DIII) by two broadly neutralizing antibodies (bNAbs), 4E11 and 4E5A. There are four serotypes of Dengue virus (DENV-1 to -4), whose DIII sequences vary by up to 49%. We used combinatorial alanine scanning mutagenesis, a phage display approach, to map functional epitopes (those residues that contribute most significantly to the energetics of antibody-antigen interaction) on these four serotypes. Our results showed that 4E11, which binds strongly to DENV-1, -2, and -3, and moderately to DENV-4, recognized a common conserved core functional epitope involving DIII residues K310, L/I387, L389, and W391. There were also unique recognition features for each serotype, suggesting that 4E11 has flexible recognition requirements. Similar scanning studies for the related bNAb 4E5A, which binds more tightly to DENV-4, identified broader functional epitopes on DENV-1. These results provide useful information for immunogen and therapeutic antibody design.

Temperature-dependent folding allows stable dimerization of secretory and virus-associated E proteins of Dengue and Zika viruses in mammalian cells

“…Mouse mAb 4E5A 40 was kindly provided by Prof. Jonathan R. Lai (Albert Einstein College of Medicine, NY, USA). mAbs EDE1-C8 (clone 752-2 C8), EDE1-C10 (clone 753(3) C10) and EDE2-B7 (clone 747 B7) were constructed as human IgG antibodies from previously deposited sequences 27 and used as culture supernatants from transfected cells.…”

Protein and Antibody Engineering by Phage Display

“…In our lab, phagemid HP153 has been shown to readily display many different proteins including domain III (DIII) of the glycoprotein E of DENV (Frei et al, 2015), Sudan virus (SUDV) Fabs (Chen et al, 2014), Dengue Fab E106 (unpublished results), HIV-1 Fab 2G12 (Stewart, Liu, & Lai, 2012), and protein G domain B1 from Streptococcus (unpublished results). To detect the protein of interest on phage and to monitor expression levels, we include a FLAG epitope (DYKDDDDK) either at the N-terminus (Dengue DIII, protein G) or C-terminus (light chain of Fabs), and probe for it using the anti-FLAG antibody M2.…”

“…However, in other cases where one seeks to engineer new function into a scaffold or engineer an alternative binding interface, several sublibraries or a semiempirical step-wise approach may be required to identify which residues should be varied (Koide, Wojcik, Gilbreth, Hoey, & Koide, 2012; Mandal et al, 2012). Our lab has successfully used phage display to humanize an anti-SUDV mouse antibody (Chen et al, 2014), isolate antibodies specific to different proteolytic intermediates of the Ebola glycoprotein (Koellhoffer et al, 2012), isolate Fabs against proapoptosis protein BAX (Uchime et al, 2016), and map the hotspot residues in antibody–antigen interactions on both the antigen (DENV DIII) or the antibody (Da Silva, Harrison, & Lai, 2010; Frei et al, 2015; Liu, Regula, Stewart, & Lai, 2011; Stewart, Harrison, Regula, & Lai, 2013; Stewart et al, 2012). …”

“…This technique readily identifies “hotspot residues,” or residues that contribute substantial binding energy to the interaction. Combinatorial alanine scanning with phage display has been used to probe the energetics of the growth hormone and growth hormone receptor interface, as well as, to characterize residues that contribute substantially to the binding of a Dengue virus (DENV) broadly neutralizing antibody to its antigens (Frei, Kielian, & Lai, 2015; Pal, Kossiakoff, & Sidhu, 2003; Weiss, Watanabe, Zhong, Goddard, & Sidhu, 2000). We describe here how to design, synthesize, and screen phage libraries, including a list of troubleshooting steps for common problems that arise during the phage display process.…”

Preface

“…For this reason, advances for employing non-coded or even non-natural amino acids are discussed (Coin et al 2011; Hu et al 2014). Additionally, libraries of antibodies or other proteins are often made as libraries so methods are needed to display these products (Frei and Lei 2015). …”