Thesis

Abstract

Rational engineering of multimeric constructs for ultra-sensitive point-of-care diagnostics against Ebola secreted glycoprotein

Ebolavirus (EBOV) hemorrhagic fever outbreaks post serious public health emergencies due to EBOV disease ease of contagion and high mortality rates. EBOV outbreaks are difficult to predict and contain due to the zoonotic nature of this family of viruses. The need for rapid isolation and early treatment of those exposed warrants the development of highly sensitive and specific point-of-care tests (POCT) capable of screening at-risk population in low-resource settings. Traditionally, POCTs rely on high affinity antibody pairs; however, generating these pairs is costly and challenging. Here we show that yeast display of synthetic nanobodies (Nb) and rational engineering of Nb multimers may present a more efficient and cost-effective method to developing high-performing POCTs. Targeting EBOV secreted glycoprotein, we screened a 10⁷-sized synthetic Nb library and identified an overall promising clone with a K_D of 200 nM. To enhance the binding off-rate, we used a hypermutating modality of yeast display to evolve and affinity mature our most promising Nb, attaining a 10-fold improvement in K_D. Using the avidity effect to further advantage these Nbs, we utilized CryoEM and thermodynamic modeling to rationally engineer linkers, yielding multimeric Nbs with sub-nanomolar affinity. The strategies explored here broadly propose streamlined methodologies for synthetic binder development. More specifically, our work offers a novel pipeline to rapidly developing ultrasensitive POCTs, contributing to means for fast containment and effective early treatment of EBOV and other similarly contagious and deadly diseases in low-resource settings.

My thesis is available upon email request: Joanna.peng@duke.edu