Structural and antigenic characterization of novel and diverse Henipavirus glycoproteins

Henipaviruses (HNVs), a genus within the Paramyxoviridae family, includes the highly virulent Nipah and Hendra viruses that cause yearly reoccurring outbreaks of deadly disease. Recent discoveries of several new Henipavirus species, including the zoonotic Langya virus, have revealed much higher antigenic diversity than currently characterized. Here, to explore the limits of structural and antigenic variation in HNVs, we construct an expanded, antigenically diverse panel of HNV fusion (F) and attachment (G) glycoproteins from 56 unique HNV strains that better reflects global HNV diversity. We expressed and purified the F ectodomains and the G head domains, characterized their biochemical, biophysical and structural properties. We performed immunization experiments in mice leading to the elicitation of antibodies reactive to multiple HNV F proteins. Cryo-EM structures of diverse F proteins elucidate molecular determinants of differential pre-fusion state metastability and higher order contacts. A crystal structure of the Gamak virus G head domain revealed an additional domain added to the conserved 6-bladed, β-propeller fold. Taken together, these studies expand the known structural and antigenic limits of the Henipavirus genus, reveal new cross-reactive epitopes within the HNV genus and provide foundational data needed for the development of broadly reactive countermeasures.