Direct and indirect interactions in the recognition between a cross‐neutralizing antibody and the four serotypes of dengue virus

Abstract

Dengue fever is the most important vector-borne viral disease. Four serotypes of dengue virus, DENV1 to DENV4, coexist. Secondary infection by a different serotype is a risk factor for severe dengue. Monoclonal antibody mAb4E11 neutralizes the four serotypes of DENV with varying efficacies by recognizing an epitope located within domain-III (ED3) of the viral envelope (E) protein. To better understand the cross-reactivities between mAb4E11 and the four serotypes of DENV, we constructed mutations in both Fab4E11 fragment and ED3, and we searched for indirect interactions in the crystal structures of the four complexes. According to the serotype, 7 to 12 interactions are mediated by one water molecule, 1 to 10 by two water molecules, and several of these interactions are conserved between serotypes. Most interfacial water molecules make hydrogen bonds with both antibody and antigen. Some residues or atomic groups are engaged in both direct and water-mediated interactions. The doubly-indirect interactions are more numerous in the complex of lowest affinity. The third complementarity determining region of the light chain (L-CDR3) of mAb4E11 does not contact ED3. The structures and double-mutant thermodynamic cycles showed that the effects of (hyper)-mutations in L-CDR3 on affinity were caused by conformational changes and indirect interactions with ED3 through other CDRs. Exchanges of residues between ED3 serotypes showed that their effects on affinity were context dependent. Thus, conformational changes, structural context, and indirect interactions should be included when studying cross-reactivity between antibodies and different serotypes of viral antigens for a better design of diagnostics, vaccine, and therapeutic tools against DENV and other Flaviviruses.

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

“…Previously reported site-directed mutagenesis studies on this interaction focused only on a single serotype (DENV-1) and a subset of residues (Bedouelle et al, 2006; Lisova et al, 2014; Lisova et al, 2007). In addition, an important aspect of combinatorial alanine scanning is benchmarking to previous site-directed mutagenesis studies when available.…”

“…For example, the ΔΔG Ala-WT values from combinatorial alanine scanning and traditional site-directed mutagenesis studies correlate closely in the case of hGH-hGHR, and to lesser extents in other systems (Clackson and Wells, 1995; Da Silva et al, 2010; Pál et al, 2005; Weiss et al, 2000). We therefore compared our alanine scanning results with previously published mutagenesis data by Bedouelle and coworkers for DENV-1 DIII in which individual DIII alanine mutants were purified and tested for their binding affinities for 4E11 (Bedouelle et al, 2006; Lisova et al, 2014; Lisova et al, 2007). The Bedouelle study encompassed 14 total A/G strand residues, 11 of which overlapped with our subset, and three of which are not included in our analysis.…”

“…In several viral systems including Dengue, HIV-1, and influenza, a wealth of structural information on bNAbglycoprotein complexes provides some insight into the requisite features of the structural epitopes. However, less effort has been devoted toward understanding the functional features (i.e., epitope residues that contribute most significantly to the energy of the bNAb-glycoprotein interaction) by mutagenesis studies (Bedouelle et al, 2006; Lisova et al, 2014; Lisova et al, 2007). …”

“…Here, we obtained comprehensive insight into functional epitope requirements for recognition of Dengue virus (DENV) envelope glycoprotein E domain III (DIII) by two related broadly neutralizing antibodies, 4E11 and 4E5A, as a model system (Figure 1) (Bedouelle et al, 2006; Cockburn et al, 2012a; Lisova et al, 2014; Lisova et al, 2007; Tharakaraman et al, 2013). There are four co-circulating serotypes of Dengue virus (DENV-1, -2, -3, and -4), a mosquito-spread flavivirus that causes significant disease in endemic subtropical regions (Dengue Shock Syndrome, DSS, and Dengue Hemorrhagic Fever, DHF) (Gubler et al, 2007; Halstead and O’rourke, 1977; Sangkawibha et al, 1984).…”

Characterization and epitope mapping of Dengue virus type 1 specific monoclonal antibodies

“…Binding of antibodies to EDIII domain have been suggested to alter the virus structure, trap the antigen in an already existing conformation, or induce molecular rearrangements prior to epitope binding [ 32 , 44 , 45 ]. EDIII is an immunoglobulin-like module and putatively contains epitopes for neutralizing antibodies and the receptor-binding site [ 46 – 48 ]. Investigating the 3-D structure of the E protein reveals that the Thr 346 and Asp 360 residues are indeed spatially exposed on the surface and accessible in the transition states.…”