Diversity and junction residues as hotspots of binding energy in an antibody neutralizing the dengue virus

Abstract

Dengue is a disease which is re-emerging, viral and transmitted by the Aedes mosquitoes. Approximately 100 million individuals are affected by the disease annually and one billion are at risk, mainly in the subtropical regions. Severe forms of the disease can lead to death within hours. There is an urgent need for preventive or curative tools to fight against the dengue virus, because no such specific treatment exists to date.The virus has four serotypes, DEN1 to DEN4. Several tetravalent vaccines are under development but they will not be available for at least a decade, and comprehensive vaccinal coverage might be difficult to achieve [1,2].The dengue virus is an enveloped RNA virus. The structures of the whole virus and of its envelope glycoprotein E have been elucidated by a combination of Dengue is a re-emerging viral disease, affecting approx. 100 million individuals annually. The monoclonal antibody mAb4E11 neutralizes the four serotypes of the dengue virus, but not other flaviviruses. Its epitope is included within the highly immunogenic domain 3 of the envelope glycoprotein E. To understand the favorable properties of recognition between mAb4E11 and the virus, we recreated the genetic events that led to mAb4E11 during an immune response and performed an alanine scanning mutagenesis of its third hypervariable loops (H-CDR3 and L-CDR3). The affinities between 16 mutant Fab fragments and the viral antigen (serotype 1) were measured by a competition ELISA in solution and their kinetics of interaction by surface plasmon resonance. The diversity and junction residues of mAb4E11 (D segment; V H -D, D-J H and V L -J L junctions) constituted major hotspots of interaction energy. Two residues from the D segment (H-Trp96 and H-Glu97) provided > 85% of the free energy of interaction and were highly accessible to the solvent in a three-dimensional model of mAb4E11. Changes of residues (L-Arg90 and L-Pro95) that statistically do not participate in the contacts between antibodies and antigens but determine the structure of L-CDR3, decreased the affinity between mAb4E11 and its antigen. Changes of L-Pro95 and other neutral residues strongly decreased the rate of association, possibly by perturbing the topology of the electrostatic field of the antibody. These data will help to improve the properties of mAb4E11 for therapeutic applications and map its epitope precisely.Abbreviations -, covalent bond; ::, noncovalent bond; CDR, complementary determining region; E3, domain 3 of gpE; gpE, glycoprotein E; H-Trp96, a tryptophan residue in position 96 of the heavy chain; H-W96A, mutation of residue H-Trp96 into Ala; RU, resonance unit; SDR, structure determining residue.

Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus

“…1; see Methods). The value of K D was equal to 0.12±0.01 nM and was fully consistent with the value that we obtained previously with another recombinant form of the antigen (Bedouelle et al, 2006).To confirm the specificity of mAb4E11 for the flaviviral group of DENV, we performed indirect ELISAs of the interaction between the Fab4E11-H6 fragment and various ED3-H6 domains, i.e. those from DENV1, JEV, WNV and YFV (Table 1).…”

“…The Fab4E11-H6 fragment was produced in E. coli strain HB2151 and purified as described previously (Bedouelle et al, 2006). The ED3-H6 domains were produced in E. coli strain BL21(DE3) from recombinant plasmids ( Table 1).…”

“…Selection of peptides from a random library by the method of phage display has shown that residues 306-314 of gpE from the DENV1 serotype (gpE.DEN1) constitute a mimotope of the mAb4E11 epitope (Thullier et al, 2001). Its paratope has been partially characterized by alanine-scanning mutagenesis of its third hypervariable loops (Bedouelle et al, 2006;Renard et al, 2003). This mutagenesis study of the Fab fragment of mAb4E11 has shown that the two residues that come from its diversity segment, H-Trp96 and H-Glu97 (Kabat's numbering), together provide .85 % of the free energy of interaction with the ED3.DEN1 antigen.…”

“…Therefore, the epitope of mAb4E11 includes two basic residues, Lys307 and Lys310, and a hydrophobic pocket that could interact with the diversity residues, H-Trp96-Glu97, of mAb4E11. These two residues constitute the main hot spots of binding energy with the ED3.DEN1 antigen (Bedouelle et al, 2006). …”

“…First, the value of K D for the synthetic peptide that corresponds to residues 306-314 of gpE.DEN1 is equal to 3 mM and is thus 37 000-fold higher than the value for the whole ED3.DEN1-H6 domain (Thullier et al, 2001). Second, we have shown in a previous study that the diversity segment of mAb4E11 comprises two residues, H-Trp96 and H-Glu97; these two residues are major contributors to the interaction with ED3.DEN1 (DDG¢6 kcal mol 21 for each mutation into Ala), and they are highly exposed to the solvent in a three-dimensional model of the Fab4E11 fragment (Bedouelle et al, 2006). Thus, the epitope of mAb4E11 should contain both positively charged residues to interact with the negatively charged H-Glu97, and a hydrophobic pocket to bind H-Trp96.…”