Measures to prevent and treat Nipah virus disease: research priorities for 2024-29.

Nipah virus causes highly lethal disease, with case-fatality rates ranging from 40% to 100% in recognised outbreaks. No treatments or licensed vaccines are currently available for the prevention and control of Nipah virus infection. In 2019, WHO published an advanced draft of a research and development roadmap for accelerating development of medical countermeasures, including diagnostics, therapeutics, and vaccines, to enable effective and timely emergency response to Nipah virus outbreaks. This Personal View provides an update to the WHO roadmap by defining current research priorities for development of Nipah virus medical countermeasures, based primarily on literature published in the last 5 years and consensus opinion of 15 subject matter experts with broad experience in development of medical countermeasures for Nipah virus or experience in the epidemiology, ecology, or public health control of outbreaks of Nipah virus. The research priorities are organised into four main sections: cross-cutting issues (for those that apply to more than one category of medical countermeasures), diagnostics, therapeutics, and vaccines. The strategic goals and milestones identified in each section focus on key achievements that are needed over the next 6 years to ensure that the necessary tools are available for rapid response to future outbreaks of Nipah virus or related henipaviruses.

Foals of mares vaccinated for Hendra virus have a suboptimal response to HeV vaccination.

Hendra virus (HeV) is lethal to horses and a zoonotic threat to humans in Australia, causing severe neurological and/or respiratory disease with high mortality. An equine vaccine has been available since 2012. Foals acquire antibodies from their dams by ingesting colostrum after parturition, therefore it is assumed that foals of mares vaccinated against HeV will have passive HeV antibodies circulating during the first several months of life until they are actively vaccinated. However, no studies have yet examined passive or active immunity against HeV in foals. Here, we investigated anti-HeV antibody levels in vaccinated mares and their foals. Testing for HeV neutralising antibodies is cumbersome due to the requirement for Biosafety level 4 (BSL-4) containment to conduct virus neutralisation tests (VNT). For this study, a subset of samples was tested for HeV G-specific antibodies by both an authentic VNT with infectious HeV and a microsphere-based immunoassay (MIA), revealing a strong correlation. An indicative neutralising level was then applied to the results of a larger sample set tested using the MIA. Mares had high levels of HeV-specific neutralising antibodies at the time of parturition. Foals acquired high levels of maternal antibodies which then waned to below predictive protective levels in most foals by 6 months old when vaccination commenced. Foals showed a suboptimal response to vaccination, suggesting maternal antibodies may interfere with active vaccination. The correlation analysis between the authentic HeV VNT and HeV MIA will enable further high throughput serological studies to inform optimal vaccination protocols for both broodmares and foals.

Nano-Assembled Polyphosphazene Delivery System Enables Effective Intranasal Immunization with Nipah Virus Subunit Vaccine.

The ultimate vaccine against infections caused by Nipah virus should be capable of providing protection at the respiratory tract─the most probable port of entry for this pathogen. Intranasally delivered vaccines, which target nasal-associated lymphoid tissue and induce both systemic and mucosal immunity, are attractive candidates for enabling effective vaccination against this lethal disease. Herein, the water-soluble polyphosphazene delivery vehicle assembles into nanoscale supramolecular constructs with the soluble extracellular portion of the Hendra virus attachment glycoprotein─a promising subunit vaccine antigen against both Nipah and Hendra viruses. These supramolecular constructs signal through Toll-like receptor 7/8 and promote binding interactions with mucin─an important feature of effective mucosal adjuvants. High mass contrast of phosphorus-nitrogen backbone of the polymer enables a successful visualization of nanoconstructs in their vitrified state by cryogenic electron microscopy. Here, we characterize the self-assembly of polyphosphazene macromolecule with biologically relevant ligands by asymmetric flow field flow fractionation, dynamic light scattering, fluorescence spectrophotometry, and turbidimetric titration methods. Furthermore, a polyphosphazene-enabled intranasal Nipah vaccine candidate demonstrates the ability to induce immune responses in hamsters and shows superiority in inducing total IgG and neutralizing antibodies when benchmarked against the respective clinical stage alum adjuvanted vaccine. The results highlight the potential of polyphosphazene-enabled nanoassemblies in the development of intranasal vaccines.

Serological evidence of zoonotic filovirus exposure among bushmeat hunters in Guinea.

Human Ebola virus (EBOV) outbreaks caused by persistent EBOV infection raises questions on the role of zoonotic spillover in filovirus epidemiology. To characterise filovirus zoonotic exposure, we collected cross-sectional serum samples from bushmeat hunters (n = 498) in Macenta Prefecture Guinea, adjacent to the index site of the 2013 EBOV-Makona spillover event. We identified distinct immune signatures (20/498, 4.0%) to multiple EBOV antigens (GP, NP, VP40) using stepwise ELISA and Western blot analysis and, live EBOV neutralisation (5/20; 25%). Using comparative serological data from PCR-confirmed survivors of the 2013-2016 EBOV outbreak, we demonstrated that most signatures (15/20) were not plausibly explained by prior EBOV-Makona exposure. Subsequent data-driven modelling of EBOV immunological outcomes to remote-sensing environmental data also revealed consistent associations with intact closed canopy forest. Together our findings suggest exposure to other closely related filoviruses prior to the 2013-2016 West Africa epidemic and highlight future surveillance priorities.

An in vivo BSL-2 model for henipavirus infection based on bioluminescence imaging of recombinant Cedar virus replication in mice.

Henipaviruses are enveloped single-stranded, negative-sense RNA viruses of the paramyxovirus family. Two henipaviruses, Nipah virus and Hendra virus, cause a systemic respiratory and/or neurological disease in humans and ten additional species of mammals, with a high fatality rate. Because of their highly pathogenic nature, Nipah virus and Hendra virus are categorized as BSL-4 pathogens, which limits the number and scope of translational research studies on these important human pathogens. To begin to address this limitation, we are developing a BSL-2 model of authentic henipavirus infection in mice, using the non-pathogenic henipavirus, Cedar virus. Notably, wild-type mice are highly resistant to Hendra virus and Nipah virus infection. However, previous work has shown that mice lacking expression of the type I interferon receptor (IFNAR-KO mice) are susceptible to both viruses. Here, we show that luciferase-expressing recombinant Cedar virus (rCedV-luc) is also able to replicate and establish a transient infection in IFNAR-KO mice, but not in wild-type mice. Using longitudinal bioluminescence imaging (BLI) of luciferase expression, we detected rCedV-luc replication as early as 10 h post-infection. Viral replication peaks between days 1 and 3 post-infection, and declines to levels undetectable by BLI by 7 days post-infection. Immunohistochemistry is consistent with viral infection and replication in endothelial cells and other non-immune cell types within tissue parenchyma. Serology analyses demonstrate significant IgG responses to the Cedar virus surface glycoprotein with potent neutralizing activity in IFNAR-KO mice, whereas antibody responses in wild-type animals were non-significant. Overall, these data suggest that rCedV-luc infection of IFNAR-KO mice represents a viable platform for the study of in vivo henipavirus replication, anti-henipavirus host responses and henipavirus-directed therapeutics.

Immune and behavioral correlates of protection against symptomatic post-vaccination SARS-CoV-2 infection.

Introduction: We sought to determine pre-infection correlates of protection against SARS-CoV-2 post-vaccine inzfections (PVI) acquired during the first Omicron wave in the United States. Methods: Serum and saliva samples from 176 vaccinated adults were collected from October to December of 2021, immediately before the Omicron wave, and assessed for SARS-CoV-2 Spike-specific IgG and IgA binding antibodies (bAb). Sera were also assessed for bAb using commercial assays, and for neutralization activity against several SARS-CoV-2 variants. PVI duration and severity, as well as risk and precautionary behaviors, were assessed by questionnaires. Results: Serum anti-Spike IgG levels assessed by research assay, neutralization titers against Omicron subvariants, and low home risk scores correlated with protection against PVIs after multivariable regression analysis. Commercial assays did not perform as well as research assay, likely due to their lower dynamic range. Discussion: In the 32 participants that developed PVI, anti-Spike IgG bAbs correlated with lower disease severity and shorter duration of illness.

Structure and design of Langya virus glycoprotein antigens.

Langya virus (LayV) is a recently discovered henipavirus (HNV), isolated from febrile patients in China. HNV entry into host cells is mediated by the attachment (G) and fusion (F) glycoproteins which are the main targets of neutralizing antibodies. We show here that the LayV F and G glycoproteins promote membrane fusion with human, mouse, and hamster target cells using a different, yet unknown, receptor than Nipah virus (NiV) and Hendra virus (HeV) and that NiV- and HeV-elicited monoclonal and polyclonal antibodies do not cross-react with LayV F and G. We determined cryoelectron microscopy structures of LayV F, in the prefusion and postfusion states, and of LayV G, revealing their conformational landscape and distinct antigenicity relative to NiV and HeV. We computationally designed stabilized LayV G constructs and demonstrate the generalizability of an HNV F prefusion-stabilization strategy. Our data will support the development of vaccines and therapeutics against LayV and closely related HNVs.

Therapeutic administration of a cross-reactive mAb targeting the fusion glycoprotein of Nipah virus protects nonhuman primates.

No licensed vaccines or therapies exist for patients infected with Nipah virus (NiV), although an experimental human monoclonal antibody (mAb) cross-reactive to the NiV and Hendra virus (HeV) G glycoprotein, m102.4, has been tested in a phase 1 trial and has been provided under compassionate use for both HeV and NiV exposures. NiV is a highly pathogenic zoonotic paramyxovirus causing regular outbreaks in humans and animals in South and Southeast Asia. The mortality rate of NiV infection in humans ranges from 40% to more than 90%, making it a substantial public health concern. The NiV G glycoprotein mediates host cell attachment, and the F glycoprotein facilitates membrane fusion and infection. We hypothesized that a mAb against the prefusion conformation of the F glycoprotein may confer better protection than m102.4. To test this, two potent neutralizing mAbs against NiV F protein, hu1F5 and hu12B2, were compared in a hamster model. Hu1F5 provided superior protection to hu12B2 and was selected for comparison with m102.4 for the ability to protect African green monkeys (AGMs) from a stringent NiV challenge. AGMs were exposed intranasally to the Bangladesh strain of NiV and treated 5 days after exposure with either mAb (25 milligrams per kilogram). Whereas only one of six AGMs treated with m102.4 survived until the study end point, all six AGMs treated with hu1F5 were protected. Furthermore, a reduced 10 milligrams per kilogram dose of hu1F5 also provided complete protection against NiV challenge, supporting the upcoming clinical advancement of this mAb for postexposure prophylaxis and therapy.

Antibody targeting of conserved sites of vulnerability on the SARS-CoV-2 spike receptor-binding domain.

Given the continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs), immunotherapeutics that target conserved epitopes on the spike (S) glycoprotein have therapeutic advantages. Here, we report the crystal structure of the SARS-CoV-2 S receptor-binding domain (RBD) at 1.95 Å and describe flexibility and distinct conformations of the angiotensin-converting enzyme 2 (ACE2)-binding site. We identify a set of SARS-CoV-2-reactive monoclonal antibodies (mAbs) with broad RBD cross-reactivity including SARS-CoV-2 Omicron subvariants, SARS-CoV-1, and other sarbecoviruses and determine the crystal structures of mAb-RBD complexes with Ab246 and CR3022 mAbs targeting the class IV site, WRAIR-2134, which binds the recently designated class V epitope, and WRAIR-2123, the class I ACE2-binding site. The broad reactivity of class IV and V mAbs to conserved regions of SARS-CoV-2 VoCs and other sarbecovirus provides a framework for long-term immunotherapeutic development strategies.

Sequence basis for selectivity of ephrin-B2 ligand for Eph receptors and pathogenic henipavirus G glycoproteins.

IMPORTANCE: Ephrin-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 the 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.

mAb therapy controls CNS-resident lyssavirus infection via a CD4 T cell-dependent mechanism.

Infections with rabies virus (RABV) and related lyssaviruses are uniformly fatal once virus accesses the central nervous system (CNS) and causes disease signs. Current immunotherapies are thus focused on the early, pre-symptomatic stage of disease, with the goal of peripheral neutralization of virus to prevent CNS infection. Here, we evaluated the therapeutic efficacy of F11, an anti-lyssavirus human monoclonal antibody (mAb), on established lyssavirus infections. We show that a single dose of F11 limits viral load in the brain and reverses disease signs following infection with a lethal dose of lyssavirus, even when administered after initiation of robust virus replication in the CNS. Importantly, we found that F11-dependent neutralization is not sufficient to protect animals from mortality, and a CD4 T cell-dependent adaptive immune response is required for successful control of infection. F11 significantly changes the spectrum of leukocyte populations in the brain, and the FcRγ-binding function of F11 contributes to therapeutic efficacy. Thus, mAb therapy can drive potent neutralization-independent T cell-mediated effects, even against an established CNS infection by a lethal neurotropic virus.

Genomic Surveillance of Rabies Virus in Georgian Canines.

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eliminating dog rabies in the European Union by 2020, sporadic cases of dog rabies still occur in Eastern Europe, including Georgia. To assess the genetic diversity of the strains recently circulating in Georgia, we sequenced seventy-eight RABV-positive samples from the brain tissues of rabid dogs and jackals using Illumina short-read sequencing of total RNA shotgun libraries. Seventy-seven RABV genomes were successfully assembled and annotated, with seventy-four of them reaching the coding-complete status. Phylogenetic analyses of the nucleoprotein (N) and attachment glycoprotein (G) genes placed all the assembled genomes into the Cosmopolitan clade, consistent with the Georgian origin of the samples. An amino acid alignment of the G glycoprotein ectodomain identified twelve different sequences for this domain among the samples. Only one of the ectodomain groups contained a residue change in an antigenic site, an R264H change in the G5 antigenic site. Three isolates were cultured, and these were found to be efficiently neutralized by the human monoclonal antibody A6. Overall, our data show that recently circulating RABV isolates from Georgian canines are predominantly closely related phylogroup I viruses of the Cosmopolitan clade. Current human rabies vaccines should offer protection against infection by Georgian canine RABVs. The genomes have been deposited in GenBank (accessions: OQ603609-OQ603685).

Natural killer cells and BNT162b2 mRNA vaccine reactogenicity and durability.

Introduction: Natural killer (NK) cells can both amplify and regulate immune responses to vaccination. Studies in humans and animals have observed NK cell activation within days after mRNA vaccination. In this study, we sought to determine if baseline NK cell frequencies, phenotype, or function correlate with antibody responses or inflammatory side effects induced by the Pfizer-BioNTech COVID-19 vaccine (BNT162b2). Methods: We analyzed serum and peripheral blood mononuclear cells (PBMCs) from 188 participants in the Prospective Assessment of SARS-CoV-2 Seroconversion study, an observational study evaluating immune responses in healthcare workers. Baseline serum samples and PBMCs were collected from all participants prior to any SARS-CoV-2 infection or vaccination. Spike-specific IgG antibodies were quantified at one and six months post-vaccination by microsphere-based multiplex immunoassay. NK cell frequencies and phenotypes were assessed on pre-vaccination PBMCs from all participants by multi-color flow cytometry, and on a subset of participants at time points after the 1st and 2nd doses of BNT162b2. Inflammatory side effects were assessed by structured symptom questionnaires, and baseline NK cell functionality was quantified by an in vitro killing assay on participants that reported high or low post-vaccination symptom scores. Results: Key observations include: 1) circulating NK cells exhibit evidence of activation in the week following vaccination, 2) individuals with high symptom scores after 1st vaccination had higher pre-vaccination NK cytotoxicity indices, 3) high pre-vaccination NK cell numbers were associated with lower spike-specific IgG levels six months after two BNT162b2 doses, and 4) expression of the inhibitory marker NKG2A on immature NK cells was associated with higher antibody responses 1 and 6 months post-vaccination. Discussion: These results suggest that NK cell activation by BNT162b2 vaccination may contribute to vaccine-induced inflammatory symptoms and reduce durability of vaccine-induced antibody responses.

Structure and design of Langya virus glycoprotein antigens.

Langya virus (LayV) is a recently discovered henipavirus (HNV), isolated from febrile patients in China. HNV entry into host cells is mediated by the attachment (G) and fusion (F) glycoproteins which are the main targets of neutralizing antibodies. We show here that the LayV F and G glycoproteins promote membrane fusion with human, mouse and hamster target cells using a different, yet unknown, receptor than NiV and HeV and that NiV- and HeV-elicited monoclonal and polyclonal antibodies do not cross-react with LayV F and G. We determined cryo-electron microscopy structures of LayV F, in the prefusion and postfusion states, and of LayV G, revealing previously unknown conformational landscapes and their distinct antigenicity relative to NiV and HeV. We computationally designed stabilized LayV G constructs and demonstrate the generalizability of an HNV F prefusion-stabilization strategy. Our data will support the development of vaccines and therapeutics against LayV and closely related HNVs.

Evaluating SARS-CoV-2 Saliva and Dried Blood Spot Surveillance Strategies in a Congregate Population.

The optimal approach to COVID-19 surveillance in congregate populations remains unclear. Our study at the US Naval Academy in Annapolis, Maryland, USA, assessed the concordance of antibody prevalence in longitudinally collected dried blood spots and saliva in a setting of frequent PCR-based testing. Our findings highlight the utility of salivary-based surveillance.

Recombinant Soluble Henipavirus Glycoprotein Preparation.

Henipaviruses possess two envelope glycoproteins, the attachment (G) and the fusion (F) proteins that mediate cellular entry and are the major targets of virus-neutralizing antibody responses. Recombinant expression technologies have been used to produce soluble G and F proteins (sG and sF) that retain native-like oligomeric conformations and epitopes, which are advantageous for the development and characterization of vaccines and antiviral antibody therapeutics. In addition to Hendra virus and Nipah virus tetrameric sG and trimeric sF production, we also describe the expression and purification of Cedar virus tetrameric sG and Ghana virus trimeric sF glycoproteins. These henipavirus glycoproteins were also used as immunizing antigens to generate monoclonal antibodies, and binding was demonstrated with a pan-henipavirus multiplex microsphere immunoassay.

Recombinant Cedar Virus: A Henipavirus Reverse Genetics Platform.

The isolation of Cedar virus, a nonpathogenic henipavirus that is closely related to the highly pathogenic Nipah virus and Hendra virus, provides a new platform for henipavirus experimentation and a tool to investigate biological differences among these viruses under less stringent biological containment. Here, we detail a reverse genetics system used to rescue two replication-competent, recombinant Cedar virus variants: a recombinant wild-type Cedar virus and a recombinant Cedar virus that express a green fluorescent protein from an open reading frame inserted between the phosphoprotein and matrix genes. This recombinant Cedar virus platform may be utilized to characterize the determinants of pathogenesis across the henipaviruses, investigate their receptor tropisms, and identify novel pan-henipavirus antivirals safely under biosafety level-2 conditions.

The Sequence Basis for Selectivity of Ephrin-B2 Ligand for Eph Receptors and Pathogenic Henipavirus G Glycoproteins: Selective Ephrin-B2 Decoys for Nipah and Hendra Virus.

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.

A Recombinant Chimeric Cedar Virus-Based Surrogate Neutralization Assay Platform for Pathogenic Henipaviruses.

The henipaviruses, Nipah virus (NiV), and Hendra virus (HeV) can cause fatal diseases in humans and animals, whereas Cedar virus is a nonpathogenic henipavirus. Here, using a recombinant Cedar virus (rCedV) reverse genetics platform, the fusion (F) and attachment (G) glycoprotein genes of rCedV were replaced with those of NiV-Bangladesh (NiV-B) or HeV, generating replication-competent chimeric viruses (rCedV-NiV-B and rCedV-HeV), both with and without green fluorescent protein (GFP) or luciferase protein genes. The rCedV chimeras induced a Type I interferon response and utilized only ephrin-B2 and ephrin-B3 as entry receptors compared to rCedV. The neutralizing potencies of well-characterized cross-reactive NiV/HeV F and G specific monoclonal antibodies against rCedV-NiV-B-GFP and rCedV-HeV-GFP highly correlated with measurements obtained using authentic NiV-B and HeV when tested in parallel by plaque reduction neutralization tests (PRNT). A rapid, high-throughput, and quantitative fluorescence reduction neutralization test (FRNT) using the GFP-encoding chimeras was established, and monoclonal antibody neutralization data derived by FRNT highly correlated with data derived by PRNT. The FRNT assay could also measure serum neutralization titers from henipavirus G glycoprotein immunized animals. These rCedV chimeras are an authentic henipavirus-based surrogate neutralization assay that is rapid, cost-effective, and can be utilized outside high containment.

Shark nanobodies with potent SARS-CoV-2 neutralizing activity and broad sarbecovirus reactivity.

Despite rapid and ongoing vaccine and therapeutic development, SARS-CoV-2 continues to evolve and evade, presenting a need for next-generation diverse therapeutic modalities. Here we show that nurse sharks immunized with SARS-CoV-2 recombinant receptor binding domain (RBD), RBD-ferritin (RFN), or spike protein ferritin nanoparticle (SpFN) immunogens elicit a set of new antigen receptor antibody (IgNAR) molecules that target two non-overlapping conserved epitopes on the spike RBD. Representative shark antibody variable NAR-Fc chimeras (ShAbs) targeting either of the two epitopes mediate cell-effector functions, with high affinity to all SARS-CoV-2 viral variants of concern, including the divergent Omicron strains. The ShAbs potently cross-neutralize SARS-CoV-2 WA-1, Alpha, Beta, Delta, Omicron BA.1 and BA.5, and SARS-CoV-1 pseudoviruses, and confer protection against SARS-CoV-2 challenge in the K18-hACE2 transgenic mouse model. Structural definition of the RBD-ShAb01-ShAb02 complex enabled design and production of multi-specific nanobodies with enhanced neutralization capacity, and picomolar affinity to divergent sarbecovirus clade 1a, 1b and 2 RBD molecules. These shark nanobodies represent potent immunotherapeutics both for current use, and future sarbecovirus pandemic preparation.