Abstract
Dengue is a widespread viral disease with 3.6 billion people at risk worldwide. Humanized monoclonal antibody (mAb) 513, currently undergoing clinical trials in Singapore, targets an epitope on the envelope protein domain III exposed at the surface of the viral particle. This antibody potently neutralizes all four dengue virus serotypes in a humanized mouse model that recapitulates human dengue infection, without signs of antibody-mediated enhancement of the disease. The crystal structure of single-chain variable fragment (scFv) 513 bound to the envelope protein domain III from dengue virus serotype 4 was used as a template to explore the molecular origins of the broader cross-reactivity and increased in vivo potency of mAb 513, compared to the parent murine mAb 4E11, using molecular dynamics simulations and network analyses. These two methods are a powerful complement to existing structural and binding data and detail specific interactions that underpin the differential binding of the two antibodies. We found that a Glu at position H55 (GluH55) from the second Complementarity Determining Region of the Heavy chain (CDR-H2) which corresponds to Ala in 4E11, is a major contributor to the enhancement in the interactions of mAb 513 compared to 4E11. Importantly, we also validate the importance of GluH55 using site-directed mutagenesis followed by isothermal titration calorimetry measurements.
Highlights
Dengue is a major mosquito-borne viral disease, whose prevalence recently expanded beyond the tropical and subtropical regions of the globe, with about 3.6 billion people at risk of contracting the disease1
As anticipated, the evolution and mutations observed among Dengue virus (DENV) serotypes correlate directly with the cross-neutralization efficacy of monoclonal antibody (mAb) 4E11, 1A1D-2, 9D12 and 513, all of which bind an epitope centered on the A-strand of DIII15,18,25–28
molecular dynamics (MD) simulations suggest that the broad recognition by 513 of the four DENV serotypes results from a combination of a larger surface area buried upon complexation compared to 4E11 and specific interactions with conserved functional epitope core residues consisting of Lys310, a polymorphic site 323, Leu387 and Leu/Ile389. This is in agreement with previous experimental observations that were made for the interactions of mAb 4E11 with DENV1-432
Summary
Dengue is a major mosquito-borne viral disease, whose prevalence recently expanded beyond the tropical and subtropical regions of the globe, with about 3.6 billion people at risk of contracting the disease. The Sanofi-Pasteur CYD-TDV tetravalent vaccine, which uses the yellow fever vaccine backbone, is marketed in several countries7,8 This vaccine requires three booster injections and confers an uneven protection against the various DENV serotypes, with limited protection against DENV2. It was desirable to humanize the murine mAb 4E11 and to significantly improve the neutralization capacity of this humanized antibody towards DENV3 and DENV4, while retaining high affinity towards DENV1 and DENV2 This was accomplished in two stages: [1] using a purely computational approach, an initial improved version of 4E11 named 4E5A, was engineered by introducing five affinity-enhancing mutations in three complementarity determining regions (CDRs): this subset of mutations (CDR-L1: Arg31Lys; CDR-L2: Asn57Glu, Glu59Gln, Ser60Trp; CDR-H2 Ala55Glu) were selected from a total of 87 single mutants tested and their affinity-enhancing effects were shown to be additive (see Tables S8, S9 and S10 in ref.). Validation of the computational predictions was performed using site-directed mutagenesis targeting the hotspot residue H55 from the CDR2 of mAbs 4E11 and 513, followed by isothermal titration calorimetry measurements
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