(I-334) Rational engineering of multimeric constructs for ultra-sensitive point-of-care diagnostics against Ebola secreted glycoprotein

Introduction::
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. Rapid isolation and early treatment require a low-cost, point-of-care diagnostic (POCD) to screen at-risk populations with sensitivity and specificity. Compared to the gold standard polymerase-chain-reaction (PCR) test, which is resource-and time-intensive, POCDs produce results instantly, are easy to access, and demand less equipment. However, developing accurate and sensitive POCDs rely on high affinity antibody (Ab) pairs that can be challenging and slow to generate.




Materials and Methods::
Here we show that yeast and phage display of synthetic nanobody (Nb) and affibody (Afb) libraries may present a more efficient and less expensive method to generating high-performing POCDs. Such libraries were screened against EBOV secreted glycoprotein, an ideal diagnostic target due to its prolific secretion during the early stages of infection. To capitalize on the avidity effect, we engineered multimeric constructs by rationally designing flexible linkers through predictive modeling and informed by AlphaFold for select promising Nb clones. The binding affinity of Individual clones and resultant multimeric constructs were characterized through surface plasmon resonance.




Results, Conclusions, and Discussions::
Rationally designing linkers to multimerize a promising Nb clone resulted in a 1000-fold increase in binding, achieving a K_d ~ 0.72 nM. Hypothesizing the sensitivity could be further improved by increasing multimerization, we have used phage and yeast display to screen synthetic Afbs for complementary clones. Non-cross-reactive Afb clones have demonstrated K_d as low as 20 nM but need further characterization to ensure selected Nbs and Afbs have non-overlapping epitopes, enabling complementary binding. Our study offers a novel workflow to rapidly developing ultrasensitive POCDs, which may contribute to means for fast containment and effective early treatment of EBOV and other similarly contagious and deadly diseases in low-resource settings.




Acknowledgements (Optional): :

References (Optional): : 1.World Health Organization, “Disease outbreak news; Ebola virus disease-Democratic Republic of the Congo,” September 29, 2022. [Online]. Available at: https://www.who.int/emergencies/disease-outbreak-news/item/2022-DON411 [Accessed October 29, 2022].

2.C.M. Fontes, B.D. Lipes, J. Liu, K.N. Agans, A. Yan, P. Shi, D.F. Cruz, G. Kelly, K.M. Luginbuhl, D.Y. Joh, S.L. Foster, J. Heggestad, A. Hucknall, M.H. Mikkelsen, C. F. Pieper, R.W. Horstmeyer, T.W. Geisbert, M.D. Gunn, and A. Chilkoti, “Ultrasensitive point-of-care immunosassay for secreted glycoprotein detects Ebola infection earlier than PCR,” Science Translation Medicine, vol. 13, No. 588, April 2021.

3.Kjaergaard, M., Glavina, J., & Chemes, L. B. (2021). Predicting the effect of disordered linkers on effective concentrations and avidity with the “Ceff calculator” app. Methods in enzymology (Vol. 647, pp. 145-171).