Jays, A. | bioRxiv Science

bioRxiv2026

Computational design of potent, broadly neutralizing anti-Nipah virus and Hendra virus miniproteins

The prototype members of the genus Henipavirus, Nipah virus (NiV) and Hendra virus (HeV), cause recurrent zoonotic spillovers with case fatality rates ranging from 40-90% in humans. Currently, there are no approved vaccines or therapeutics for use in humans. Neutralizing antibodies targeting the NiV/HeV F- or G-glycoproteins protect animals from lethal challenge and are a main correlate of protection. However, antibody-based formulations are expensive, typically requiring hospital admission for administration and cold-chain for storage and transportation. To address the lack of shelf-stable clinical countermeasures, we computationally designed thermostable miniproteins that cross-react with subnanomolar affinities with both NiV and HeV F and G glycoproteins and inhibit viral entry in vitro with potencies comparable to lead antibodies. Oligomerized forms of these miniproteins have enhanced potency relative to their monomeric building blocks and increase the barrier for emergence of escape mutants, establishing them as promising preclinical candidates against these deadly viruses.

bioengineering↗

bioRxiv2023

The Sequence Basis for Selectivity of Ephrin-B2 Ligand for Eph Receptors and Pathogenic Henipavirus G Glycoproteins

Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and functions as a cell entry receptor for several henipaviruses including Nipah virus (NiV), a pathogenic zoonotic virus with pandemic potential. To understand the sequence basis of promiscuity for EFNB2 binding to the attachment glycoprotein of NiV (NiV-G) and Eph receptors, we performed deep mutagenesis on EFNB2 to identify mutations that enhance binding to NiV-G over EphB2, one of the highest affinity Eph receptors. The mutations highlight how different EFNB2 conformations are selected by NiV-G versus EphB2. Specificity mutations are enriched at the base of the G-H binding loop of EFNB2, especially surrounding a phenylalanine hinge upon which the G-H loop pivots, and at a phenylalanine hook that rotates away from the EFNB2 core to engage Eph receptors. One EFNB2 mutant, D62Q, possesses pan-specificity to the attachment glycoproteins of closely related henipaviruses and has markedly diminished binding to the six Eph receptors. However, EFNB2-D62Q has high residual binding to EphB3 and EphB4. A second deep mutational scan of EFNB2 identified combinatorial mutations to further enhance specificity to NiV-G. A triple mutant of soluble EFNB2, D62Q-Q130L-V167L, has minimal binding to Eph receptors but maintains binding, albeit reduced, to NiV-G. Soluble EFNB2 decoy receptors carrying the specificity mutations were potent neutralizers of chimeric henipaviruses. These findings demonstrate how specific residue changes at the shared binding interface of a promiscuous ligand (EFNB2) can influence selectivity for multiple receptors, and may also offer insight towards the development of henipavirus therapeutics and diagnostics. IMPORTANCEEphrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and regulates multiple cell developmental and signaling processes. It also functions as the cell entry receptor for Nipah virus and Hendra virus, zoonotic viruses that can cause respiratory and/or neurological symptoms in humans with high mortality. Here, we investigate the sequence basis of EFNB2 specificity for binding the Nipah virus attachment G glycoprotein over Eph receptors. We then use this information to engineer EFNB2 as a soluble decoy receptor that specifically binds the attachment glycoproteins of Nipah virus and other related henipaviruses to neutralize infection. These findings further mechanistic understanding of protein selectivity and may facilitate the development of diagnostics or therapeutics against henipavirus infection.

microbiology↗