AUTHOR=Hruškovicová Jana , Bhide Katarína , Petroušková Patrícia , Tkáčová Zuzana , Mochnáčová Evelína , Čurlík Ján , Bhide Mangesh , Kulkarni Amod 

TITLE=Engineering the Single Domain Antibodies Targeting Receptor Binding Motifs Within the Domain III of West Nile Virus Envelope Glycoprotein

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 13 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.801466

DOI=10.3389/fmicb.2022.801466

ISSN=1664-302X

ABSTRACT=West Nile virus (WNV) is a mosquito-borne neurotrophic flavivirus causing mild febrile illness to severe encephalitis and acute flaccid paralysis with long-term or permanent neurological disorders. Due to the absence of targeted therapy or vaccines there is a growing need to develop effective anti-WNV therapy. In the present study, single domain antibodies (sdAbs) were developed against the domain III (DIII) of WNV’s envelope glycoprotein to block the entry of WNV with the human brain microvascular endothelial cells (hBMEC). Peripheral blood mononuclear cells of llama immunized with recombinant DIIIL297-S403 (rDIII) were used to generate VHH-E. coli library and phage display was performed using M13K07ΔpIII Hyperphages system. Phages displaying sdAbs against rDIII were panned with the synthetic analogs of DIII receptor binding motifs: DIII-1G299-K307 and DIII-2V371-R388 and the VHH gene from the eluted phages was subcloned into E. coli SHuffle. Soluble sdAbs purified from 96 E. coli SHuffle clones were screened to identify 20 candidates strongly binding to synthetic analogs of DIII-1G299-K307 and DIII-2V371-R388 on a Dot blot assay. Among them, sdAbA1, sdAbA6, sdAbA9, and sdAbA10 blocked the interaction between rDIII and hBMEC on western blot and on cell ELISA. However, optimum stability during the overexpression was noticed only for sdAbA10 and it also neutralized the West Nile virus-like particles (WNV-VLP) in Luciferase assay with an EC50 of 5709.86 (23 ng/ml). Furthermore, hemocompatiblity and cytotoxicity of sdAbA10 was assessed by hemolytic assay and XTT based hBMEC proliferation assay resulting in 0.1% of hemolytic activity and 82% hBMEC viability respectively. Therefore, sdAbA10 targeting DIII-2V371-R388 of WNV envelope glycoprotein is observed to be suitable for in vivo trials as a specific therapy for West Nile virus induced neuropathogenesis.