Structure and antigenicity of divergent Henipavirus fusion glycoproteins

Abstract

In August 2022, a novel henipavirus (HNV) named Langya virus (LayV) was isolated from patients with severe pneumonic disease in China. This virus is closely related to Mòjiāng virus (MojV), and both are divergent from the bat-borne HNV members, Nipah (NiV) and Hendra (HeV) viruses. The spillover of LayV is the first instance of a HNV zoonosis to humans outside of NiV and HeV, highlighting the continuing threat this genus poses to human health. In this work, we determine the prefusion structures of MojV and LayV F proteins via cryogenic electron microscopy to 2.66 and 3.37 Å, respectively. We show that despite sequence divergence from NiV, the F proteins adopt an overall similar structure but are antigenically distinct as they do not react to known antibodies or sera. Glycoproteomic analysis revealed that while LayV F is less glycosylated than NiV F, it contains a glycan that shields a site of vulnerability previously identified for NiV. These findings explain the distinct antigenic profile of LayV and MojV F, despite the extent to which they are otherwise structurally similar to NiV. Our results carry implications for broad-spectrum HNV vaccines and therapeutics, and indicate an antigenic, yet not structural, divergence from prototypical HNVs.

Introduction

Henipaviruses (HNVs) are regarded as the most lethal paramyxoviruses, with a case fatality rate of ~70%^1,2. The HNV genus was first established after identification of the two prototypical members: Nipah virus (NiV) and Hendra virus (HeV). Since their emergence in the 1990’s, numerous spillover events of HeV from bats to horses have occurred in Australia, resulting in several human exposures³. Furthermore, NiV spillovers to humans occur almost annually in Bangladesh, and several outbreaks have also been recorded in India and the Philippines³. In recent years, the HNV genus has expanded significantly, with new viruses discovered in Africa, Australia, Asia and South America. These include Cedar virus (CedV), isolated from flying foxes in Australia⁴; Ghana virus (GhV), isolated from bats in Africa⁵; Mòjiāng virus (MojV), sequenced from rats in China⁶; Gamak & Daeryong viruses, discovered in shrews in the Republic of Korea⁷, and most recently Langya virus (LayV), identified in a throat swab from a human patient in China⁸. Outside of NiV and HeV, LayV is the only other HNV that is known to infect humans, however it is suspected that MojV and GhV also possess pathogenic potential.

Henipaviral diseases are listed on the WHO list of priority diseases that require exigent research into vaccines and therapeutics. This is largely due to the high associated case fatality rate and the global migratory patterns of the Pteropus fruit bats, which act as the main animal reservoir for HNVs⁹. With the identification of MojV and LayV, the animal reservoir of HNVs has expanded to rats and shrews. Together, these factors increase the likelihood of spillover to livestock and humans. There are currently no approved vaccines for HNVs, however a HeV G vaccine is available for veterinary use and an analogous subunit vaccine is currently in clinical development for human use^10,11,12,13. Based on sequence homology between LayV and HeV (~24%), this vaccine candidate is unlikely to confer protection to divergent HNVs. As such, there is a clear need for improved vaccine preparedness against these emerging pathogens.

An alternate vaccine target to HNV G is the fusion (F) glycoprotein. The HNV F protein is a trimeric type I fusion protein that is initially expressed as an F precursor (F₀), consisting of three domains (DI, DII and DIII), a C-terminal stem, a transmembrane domain and a cytoplasmic tail. During infection, the F₀ protein is cleaved by cathepsin L protease into F₁ and F₂ subunits, which are linked by disulfide bonds and together constitute the fusogenic F protein^14,15,16. The prefusion conformation of NiV and HeV F have been determined along with the fusion core of NiV^17,18,19. These structures highlight the large conformational changes that take place within F during fusion, where two heptad repeats in DIII and the stem (HRA and HRB) coalesce into a six-helix bundle, which is required for insertion of the fusion peptide into the host cell membrane²⁰. This outlines a rational basis for neutralizing antibody discovery and structure-based vaccine design against prefusion F. Indeed, several antibodies targeting the prefusion conformation of F have been isolated and shown to be neutralizing and protective^21,22,23,24. Moreover, vaccine studies with prefusion-stabilized NiV F have been shown to elicit potent neutralizing responses^25,26,27,28. While these studies yielded vital information into HNV vaccine design, it is unknown whether this immunity extends to other divergent HNVs such as MojV and LayV.

The F and G proteins of HNV are glycoproteins, as they are post-translationally modified with oligosaccharide structures known as glycans. Two common types of glycans, N-linked or O-linked, are covalently attached to nitrogen or oxygen atoms in amino acid side chains, respectively. Protein glycosylation can alter immunity and antibody sensitivity by shielding or exposing viral protein epitopes, as has been observed for SARS-CoV-2²⁹ and HIV³⁰. Glycosylation can also impact the structures and dynamics of proteins. With respect to HNV F proteins, mutations of the sites of N-glycosylation have shown they are required for proper folding and processing of NiV and HeV F^31,32. Site-specific glycosylation of glycoproteins can be studied using mass spectrometry glycoproteomics, as has been applied to HeV G³¹. However, there are few studies investigating the glycosylation of HNV F proteins, which is likely to be important in the context of immunity and vaccine design.

Structure-based rational vaccine design has helped improve and implement several vaccine candidates, including the SARS-CoV-2 prefusion-stabilized two-proline spike antigen formulated in Pfizer and Moderna mRNA vaccines and respiratory syncytial virus (RSV) F now in late-stage clinical development^{33,34,35,36,37}. These antigens are stabilized in the prefusion conformation by a set of mutations and/or a trimerization domain, allowing for elicitation of prefusion-specific antibodies. A similar approach to that undertaken with RSV was recently shown to stabilize the NiV and HeV F glycoproteins in the prefusion conformation²⁶. In parallel, we have previously shown that prefusion NiV F can be stabilized by a novel molecular clamp trimerization domain in the absence of heterologous mutations^25,28. Here, we use a similar approach to determine the structures of LayV and MojV F glycoproteins in the prefusion conformation via cryogenic electron microscopy (cryo-EM) and to characterize their glycosylation profiles with mass spectrometry glycoproteomics, in order to inform future vaccine design and therapy against these emerging viruses.

Results

Cryo-EM structures of LayV and MojV reveal a similar architecture to NiV F despite sequence divergence

Based on the sequence of LayV, published in August 2022, we expressed the F ectodomain residues 1-478 without any heterologous stabilizing mutations (Fig. 1a)⁸. To stabilize the prefusion form, we added a proprietary molecular clamp (clamp2) domain, analogous to that previously described^25,28,38, to the C-terminus of F. This construct was transfected into ExpiCHO-S cells, which yielded ~5.3 mg/L of recombinant prefusion-stabilized F. Protein purification from cell culture supernatant was made possible by immunoaffinity with a monoclonal antibody made in-house targeting the proprietary clamp2 domain. Prefusion LayV F proteins displayed the expected monomeric molecular weight of ~66 kDa on SDS-PAGE under reducing conditions and were devoid of contaminants (Fig. 1b). LayV F was further purified to homogeneity by size-exclusion chromatography (SEC), where a major trimeric peak was observed (Fig. 1c). In contrast, unstabilized LayV F proteins displayed a SEC profile with both trimer and aggregate peaks present (Fig S1), likely due to exposure of the hydrophobic fusion peptide leading to aggregation as well as lack of stabilization leading to dissociation of F₁ and F₂ subunits. Interestingly, unstabilized MojV F prepared via an analogous method appeared trimeric on SEC, however further investigation by negative stain electron microscopy revealed a typical postfusion conformation (Fig. S1), consistent with previous reports of unstabilized HNV F proteins³⁹. We found that addition of the clamp2 domain aided in stabilizing the prefusion form. Indeed, 2D class averages generated from initial cryo-EM images revealed particles resembling the canonical three-fold symmetry HNV F protein structure (Fig. S1). Concurrently, we applied the same methodology to MojV F, where ectodomain residues 1–482 were used to express the prefusion conformation (Fig. S2)….