Structure and antigenicity of divergent Henipavirus fusion glycoproteins.

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 Mòjiang 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.

A platform technology for generating subunit vaccines against diverse viral pathogens.

The COVID-19 pandemic response has shown how vaccine platform technologies can be used to rapidly and effectively counteract a novel emerging infectious disease. The speed of development for mRNA and vector-based vaccines outpaced those of subunit vaccines, however, subunit vaccines can offer advantages in terms of safety and stability. Here we describe a subunit vaccine platform technology, the molecular clamp, in application to four viruses from divergent taxonomic families: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), Lassa virus (LASV) and Nipah virus (NiV). The clamp streamlines subunit antigen production by both stabilising the immunologically important prefusion epitopes of trimeric viral fusion proteins while enabling purification without target-specific reagents by acting as an affinity tag. Conformations for each viral antigen were confirmed by monoclonal antibody binding, size exclusion chromatography and electron microscopy. Notably, all four antigens tested remained stable over four weeks of incubation at 40°C. Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge.

A unified route for flavivirus structures uncovers essential pocket factors conserved across pathogenic viruses.

The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.

Implications of Dengue Virus Maturation on Vaccine Induced Humoral Immunity in Mice.

The use of dengue virus (DENV) vaccines has been hindered by the complexities of antibody dependent enhancement (ADE). Current late-stage vaccine candidates utilize attenuated and chimeric DENVs that produce particles of varying maturities. Antibodies that are elicited by preferentially exposed epitopes on immature virions have been linked to increased ADE. We aimed to further understand the humoral immunity promoted by DENV particles of varying maturities in an AG129 mouse model using a chimeric insect specific vaccine candidate, bDENV-2. We immunized mice with mature, partially mature, and immature bDENV-2 and found that immunization with partially mature bDENV-2 produced more robust and cross-neutralizing immune responses than immunization with immature or mature bDENV-2. Upon challenge with mouse adapted DENV-2 (D220), we observed 80% protection for mature bDENV-2 vaccinated mice and 100% for immature and partially mature vaccinated mice, suggesting that protection to homotypic challenge is not dependent on maturation. Finally, we found reduced in vitro ADE at subneutralising serum concentrations for mice immunized with mature bDENV-2. These results suggest that both immature and mature DENV particles play a role in homotypic protection; however, the increased risk of in vitro ADE from immature particles indicates potential safety benefits from mature DENV-based vaccines.

A chimeric dengue virus vaccine candidate delivered by high density microarray patches protects against infection in mice.

Dengue viruses (DENV) cause an estimated 390 million infections globally. With no dengue-specific therapeutic treatment currently available, vaccination is the most promising strategy for its control. A wide range of DENV vaccines are in development, with one having already been licensed, albeit with limited distribution. We investigated the immunogenicity and protective efficacy of a chimeric virus vaccine candidate based on the insect-specific flavivirus, Binjari virus (BinJV), displaying the structural prM/E proteins of DENV (BinJ/DENV2-prME). In this study, we immunized AG129 mice with BinJ/DENV2-prME via a needle-free, high-density microarray patch (HD-MAP) delivery system. Immunization with a single, 1 µg dose of BinJ/DENV2-prME delivered via the HD-MAPs resulted in enhanced kinetics of neutralizing antibody induction when compared to needle delivery and complete protection against mortality upon virus challenge in the AG129 DENV mouse model.

A broadly protective antibody that targets the flavivirus NS1 protein.

There are no approved flaviviral therapies and the development of vaccines against flaviruses has the potential of being undermined by antibody-dependent enhancement (ADE). The flavivirus nonstructural protein 1 (NS1) is a promising vaccine antigen with low ADE risk but has yet to be explored as a broad-spectrum therapeutic antibody target. Here, we provide the structural basis of NS1 antibody cross-reactivity through cocrystallization of the antibody 1G5.3 with NS1 proteins from dengue and Zika viruses. The 1G5.3 antibody blocks multi-flavivirus NS1-mediated cell permeability in disease-relevant cell lines, and therapeutic application of 1G5.3 reduces viremia and improves survival in dengue, Zika, and West Nile virus murine models. Finally, we demonstrate that 1G5.3 protection is independent of effector function, identifying the 1G5.3 epitope as a key site for broad-spectrum antiviral development.