Structure, function, and antigenicity of emerging henipavirus surface glycoproteins

Abstract Henipavirus (HNV) genus, named after the first two identified members, Hendra virus (HeV) and Nipah virus (NiV), is a group of expanding zoonotic viruses that have caused repeated outbreaks with case fatality rate reaching 75%. The exceptional broad species tropism and various transmission routes make HNV a risk of potential future pandemics. Both HeV and NiV have been categorized as Biological Safety Level-4 (BSL-4) transboundary agents, and NIAID Category C Priority Pathogens. There are six emerging HNVs reported in recent years, showing limited antigenic cross-reactivity with NiV and HeV. Glycoproteins F and G are the two only spikes on the HNV surface, which coordinate the viral entry process via G glycoprotein-mediated receptor attachment followed by the F glycoprotein-mediated membrane fusion between virus and host cell. Both G and F proteins are the targets of HNV-neutralizing antibodies. Vaccine and monoclonal antibody (mAb) countermeasure development focusing on these two HNV glycoproteins are now of critical and urgent importance to prepare for potential HNV spillover events. The ectodomain of HNV G glycoprotein is a homo- tetramer with each protomer composed of a globular head and a stalk region. The metastable tetrameric conformation has restricted the previous structural characterization of G protein to the monomeric head only, which is a major obstacle for a comprehensive understanding of the G protein-mediated mechanisms of HNV entry and antibody recognition. HNV F glycoprotein trimer transits from the prefusion to the post-fusion conformation during viral entry process. The metastable prefusion conformation can be preferably recognized by neutralizing antibodies, whereby a promising target for vaccine design and therapeutic development. While the glycoproteins of both HeV and NiV have been extensively studied, the glycoproteins of the emerging HNVs, which are genetically and antigenically distinct from the two prototypic HNVs, also extremely diverse among themselves, have not been well investigated. We have used rational designs to created soluble, stabilized, oligomeric glycoprotein ectodomain constructs from several emerging HNVs, including two phylogenically distant bat-borne HNVs, CedPV and AngV, to facilitate structural, functional and antigenicity characterization. We propose to use structural and structure-based functional analyses of these glycoproteins to assist delineation of the function of HNV glycoprotein-mediated entry. We will also define the antigenicity of these HNV glycoproteins with neutralizing antibodies, including conventional murine antibodies and camelid single-domain antibodies, to investigate their neutralization epitopes and mechanisms structurally and functionally. The combined antigenicity results will render a comprehensive definition of sites of vulnerability on both G and F glycoproteins of the emerging HNVs. Collectively, findings derived from this project will provide insights to HNV entry mechanism, as well as inform future work on designing envelope glycoprotein-based vaccine and immunotherapeutic against emerging HNVs.