Molecular basis for dengue virus broad cross-neutralization by humanized monoclonal antibody 513

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.

A chimeric dengue virus vaccine candidate delivered by high density microarray patches protects against infection in mice

“…Plates were then washed 3× with PBS–0.05% Tween-20 (PBS-T). DENV antibody humanized 513 39 , conjugated with horse-radish peroxidase (HRP, Abcam) according to the manufacturer’s instructions, was added and incubated at 37 °C for 1 h. Plates were then washed 6× with PBS-T and 50 µL/well of Tetramethylbenzidine-w (TMBW, Biofx) was added. Relative absorbance was read at 450 nm on a plate reader.…”

Developing a Stabilizing Formulation of a Live Chimeric Dengue Virus Vaccine Dry Coated on a High-Density Microarray Patch

“…Fifty microliters/well of the reconstituted samples and the standard curve were added to the ELISA plate and incubated at 37 • C for one hour. Fifty microliters of humanized 513 DENV antibody [32] (h513), conjugated with horse-radish peroxidase (HRP), was incubated for one hour at 37 • C. Following incubation, 6× PBS-T washes were performed, and plates were visualized by adding tetramethylbenzidine-w (TMBW, BioFX, Eden Praire, MN, USA). The reaction was stopped by the addition of 1 M phosphoric acid, and the plate was read at 450 nm on a Varioskan LUX multimode plate reader (Thermo Fisher Scientific, Waltham, MA, USA).…”

Disease Resurgence, Production Capability Issues and Safety Concerns in the Context of an Aging Population: Is There a Need for a New Yellow Fever Vaccine?

“…One example of a promising, first-in-class monoclonal antibody for YF is TY014 developed by Tychan, Singapore [67]. TY014 was designed to neutralize multiple strains of YFV by binding to an epitope on the E protein on the surface of the virus, thereby preventing viral fusion to the host cell during viral entry [68]. TY014 will be evaluated in a Phase I clinical trial in healthy adult subjects to assess safety and the effect of TY014 on vaccine-associated viremia [67].…”