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.
Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type ...2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.
Various chemical adjuvants are available to augment immune responses to non-replicative, subunit vaccines. Optimized adjuvant selection can ensure that vaccine-induced immune responses protect ...against the diversity of pathogen-associated infection routes, mechanisms of infectious spread, and pathways of immune evasion. In this study, we compare the immune response of mice to a subunit vaccine of Middle Eastern respiratory syndrome coronavirus (MERS-CoV) spike protein, stabilized in its prefusion conformation by a proprietary molecular clamp (MERS SClamp) alone or formulated with one of six adjuvants: either (
i
) aluminium hydroxide, (
ii
) SWE, a squalene-in-water emulsion, (
iii
) SQ, a squalene-in-water emulsion containing QS21 saponin, (
iv
) SMQ, a squalene-in-water emulsion containing QS21 and a synthetic toll-like receptor 4 (TLR4) agonist 3D-6-acyl Phosphorylated HexaAcyl Disaccharide (3D6AP); (
v
) LQ, neutral liposomes containing cholesterol, 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and QS21, (
vi
) or LMQ, neutral liposomes containing cholesterol, DOPC, QS21, and 3D6AP. All adjuvanted formulations induced elevated antibody titers which where greatest for QS21-containing formulations. These had elevated neutralization capacity and induced higher frequencies of IFN
Ɣ
and IL-2-producing CD4
+
and CD8
+
T cells. Additionally, LMQ-containing formulations skewed the antibody response towards IgG2b/c isotypes, allowing for antibody-dependent cellular cytotoxicity. This study highlights the utility of side-by-side adjuvant comparisons in vaccine development.
Prior to 2020, the threat of a novel viral pandemic was omnipresent but largely ignored. Just 12 months prior to the Coronavirus disease 2019 (COVID-19) pandemic our team received funding from the ...Coalition for Epidemic Preparedness Innovations (CEPI) to establish and validate a rapid response pipeline for subunit vaccine development based on our proprietary Molecular Clamp platform. Throughout the course of 2019 we conducted two mock tests of our system for rapid antigen production against two potential, emerging viral pathogens, Achimota paramyxovirus and Wenzhou mammarenavirus. For each virus we expressed a small panel of recombinant variants of the membrane fusion protein and screened for expression level, product homogeneity, and the presence of the expected trimeric pre-fusion conformation. Lessons learned from this exercise paved the way for our response to COVID-19, for which our candidate antigen is currently in phase I clinical trial.
Nipah virus (NiV) and respiratory syncytial virus (RSV) possess two surface glycoproteins involved in cellular attachment and membrane fusion, both of which are potential targets for vaccines. The ...majority of vaccine development is focused on the attachment (G) protein of NiV, which is the immunodominant target. In contrast, the fusion (F) protein of RSV is the main target in vaccine development. Despite this, neutralising epitopes have been described in NiV F and RSV G, making them alternate targets for vaccine design. Through rational design, we have developed a vaccine strategy applicable to phylogenetically divergent NiV and RSV that comprises both the F and G proteins (FxG). In a mouse immunization model, we found that NiV FxG elicited an improved immune response capable of neutralising pseudotyped NiV and a NiV mutant that is able to escape neutralisation by two known F-specific antibodies. RSV FxG elicited an immune response against both F and G and was able to neutralise RSV; however, this was inferior to the immune response of F alone. Despite this, RSV FxG elicited a response against a known protective epitope within G that is conserved across RSV A and B subgroups, which may provide additional protection in vivo. We conclude that inclusion of F and G antigens within a single design provides a streamlined subunit vaccine strategy against both emerging and established pathogens, with the potential for broader protection against NiV.
Since the start of the COVID-19 pandemic, multiple waves of SARS-CoV-2 variants have emerged. Of particular concern is the omicron variant, which harbors 28 mutations in the spike glycoprotein ...receptor binding and N-terminal domains relative to the ancestral strain. The high mutability of SARS-CoV-2 therefore poses significant hurdles for development of universal assays that rely on spike-specific immune detection. To address this, more conserved viral antigens need to be targeted. In this work, we comprehensively demonstrate the use of nucleocapsid (N)-specific detection across several assays using previously described nanobodies C2 and E2. We show that these nanobodies are highly sensitive and can detect divergent SARS-CoV-2 ancestral, delta and omicron variants across several assays. By comparison, spike-specific antibodies S309 and CR3022 only disparately detect SARS-CoV-2 variant targets. As such, we conclude that N-specific detection could provide a standardized universal target for detection of current and emerging SARS-CoV-2 variants of concern.
The global coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spawned an ongoing demand for new research reagents and ...interventions. Herein we describe a panel of monoclonal antibodies raised against SARS-CoV-2. One antibody showed excellent utility for immunohistochemistry, clearly staining infected cells in formalin-fixed and paraffin embedded lungs and brains of mice infected with the original and the omicron variants of SARS-CoV-2. We demonstrate the reactivity to multiple variants of concern using ELISAs and describe the use of the antibodies in indirect immunofluorescence assays, Western blots, and rapid antigen tests. Finally, we illustrate the ability of two antibodies to reduce significantly viral tissue titers in K18-hACE2 transgenic mice infected with the original and an omicron isolate of SARS-CoV-2.
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.
Nipah virus (NiV) is an emergent pathogen capable of causing acute respiratory illness and fatal encephalitis in pigs and humans. A high fatality rate and broad host tropism makes NiV a serious ...public and animal health concern. There is therefore an urgent need for a NiV vaccines to protect animals and humans. In this study we investigated the immunogenicity of bovine herpesvirus (BoHV-4) vectors expressing either NiV attachment (G) or fusion (F) glycoproteins, BoHV-4-A-CMV-NiV-GΔTK or BoHV-4-A-CMV-NiV-FΔTK, respectively in pigs. The vaccines were benchmarked against a canarypox (ALVAC) vector expressing NiV G, previously demonstrated to induce protective immunity in pigs. Both BoHV-4 vectors induced robust antigen-specific antibody responses. BoHV-4-A-CMV-NiV-GΔTK stimulated NiV-neutralizing antibody titers comparable to ALVAC NiV G and greater than those induced by BoHV-4-A-CMV-NiV-FΔTK. In contrast, only BoHV-4-A-CMV-NiV-FΔTK immunized pigs had antibodies capable of significantly neutralizing NiV G and F-mediated cell fusion. All three vectored vaccines evoked antigen-specific CD4 and CD8 T cell responses, which were particularly strong in BoHV-4-A-CMV-NiV-GΔTK immunized pigs and to a lesser extent BoHV-4-A-CMV-NiV-FΔTK. These findings emphasize the potential of BoHV-4 vectors for inducing antibody and cell-mediated immunity in pigs and provide a solid basis for the further evaluation of these vectored NiV vaccine candidates.
Subunit vaccines exhibit favorable safety and immunogenicity profiles and can be designed to mimic native antigen structures. However, pairing with an appropriate adjuvant is imperative in order to ...elicit effective humoral and cellular immune responses. In this study, we aimed to determine an optimal adjuvant pairing with the prefusion form of influenza haemagglutinin (HA) or respiratory syncytial virus (RSV) fusion (F) subunit vaccines in BALB/c mice in order to inform future subunit vaccine adjuvant selection. We tested a panel of adjuvants, including aluminum hydroxide (alhydrogel), QS21, Addavax, Addavax with QS21 (AdQS21), and Army Liposome Formulation 55 with monophosphoryl lipid A and QS21 (ALF55). We found that all adjuvants elicited robust humoral responses in comparison to placebo, with the induction of potent neutralizing antibodies observed in all adjuvanted groups against influenza and in AdQS21, alhydrogel, and ALF55 against RSV. Upon HA vaccination, we observed that none of the adjuvants were able to significantly increase the frequency of CD4
and CD8
IFN-γ
cells when compared to unadjuvanted antigen. The varying responses to antigens with each adjuvant highlights that those adjuvants most suited for pairing purposes can vary depending on the antigen used and/or the desired immune response. We therefore suggest that an adjuvant trial for different subunit vaccines in development would likely be necessary in preclinical studies.
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