Functional assays for the evaluation of nanobodies targeting Henipavirus glycoproteins

Henipavirus (HNV), a genus of negative-strand RNA viruses in the Paramyxoviridae family, includesthe Hendra (HeV) and Nipah (NiV) viruses. These viruses, carried by fruit bats, can causesevere disease in humans and are classified as Biosafety level 4 pathogens due to their epidemicpotential. HeV and NiV enter host cells via two glycoproteins: the attachment (G) protein and thefusion (F) protein. The F protein undergoes a critical conformational change to facilitate viralentry, making the prefusion conformation a key target for preventing infection. Single-domainantibodies derived from camelid species, known as nanobodies, offer key advantages over conventionalantibodies for targeting glycoproteins. With this project, I aim to develop and optimizehigh-throughput functional assays to characterize nanobodies targeting the attachment (G) andfusion (F) glycoproteins of the Nipah virus (NiV) and Hendra virus (HeV). The F protein wasstabilized in the prefusion conformation with known amino acid substitutions, then cloned, expressed,and purified using Strep-tag affinity and size-exclusion chromatography. Nanobodiestargeting the glycoproteins of HeV and NiV were produced, and their binding to the glycoproteinswas investigated using ELISA. A lentivirus-based neutralization assay was set up to evaluate thenanobodies ability to neutralize HNV infection, and a dual-split protein fusion inhibition assay wasused to determine the nanobodies ability to inhibit fusion. In this project, I produced stabilizedF proteins and confirmed prefusion conformation by cryo-EM. I used these stabilized F proteinsto determine the conformation specificity of HeV and NiV F-specific nanobodies. Finally, I conductedfunctional assays to characterize the nanobodies ability to neutralize the virus and inhibitglycoprotein function, with additional refinements needed before using the assays to characterizethe nanobodies. Overall, this work paves the way for systematic characterization of nanobodylibraries targeting henipavirus glycoproteins with strong implications for vaccine design and thedevelopment of antiviral agents.