Biology subjects

Sherry, L.

Publications and source records attributed to Sherry, L..

16 recordsLinked to original sources

bioRxiv2026

Feline calicivirus encoding NanoLuc luciferase as a tool for assessing antibody neutralisation and antivirals

Feline calicivirus (FCV) is among the most common viruses to infect cats worldwide, with prevalence estimated to range from 10-90% depending on the population sampled. Typical FCV infection presents with oral ulcerations, fever and in some cases can also lead to clinical signs such as pneumonia or "limping syndrome". However, some FCV strains have been isolated from cats exhibiting virulent systemic (VS) disease, which is associated with high morbidity and mortality. Breakthrough VS-FCV infections have been recorded in vaccinated cats and, therefore, there is considerable interest in developing novel therapeutics for use in the face of VS-FCV outbreaks. However, to design effective therapeutics, a tractable system to systematically assess the efficacy of novel vaccine candidates or antivirals is required. Here, we used reverse genetics to develop an FCV reporter virus, inserting NanoLuc luciferase into the LC protein of FCV-Urbana (FCV-UrbanaNL). We characterised the replication kinetics of FCV-UrbanaNL in comparison to its parent virus and assessed the stability of the reporter over multiple passages. Subsequently, we developed virus neutralisation assays to assess a range of monoclonal antibodies that recognise FCV Urbana. We then assessed the breadth of neutralisation by exchanging the major capsid protein, VP1, of FCV Urbana with VP1 from the vaccine strain F9 and the VS-FCV strain NSW-E1. Finally, we evaluated the utility of the FCVNL reporter system to screen candidate antiviral compounds, identifying GS-441524 (the active metabolite of the parent nucleoside remdesivir) as having therapeutic potential against FCV. These findings highlight the potential of this reporter virus as a powerful molecular tool to accelerate the discovery and development of novel therapeutics.

microbiology↗

bioRxiv2026

Enhanced inter-chain hydrogen bonding in the murine norovirus VP1 capsid leads to increased particle stability and delayed viral uncoating

Capsid stability is vital for virion survival as the capsid must withstand varying environmental challenges such as pH and temperature to allow the virus to reach a target cell. Noroviruses are non-enveloped, icosahedral, positive-sense RNA viruses of importance to human health globally, with no approved vaccine or antiviral available. Despite this, the molecular mechanisms behind norovirus capsid stability and capsid rearrangement prior to RNA translocation are understudied. Using murine norovirus as a model, we utilised thermal stress to create a thermally stable virus population. By introducing three identified substitutions in the major capsid protein VP1 from this virus population into an infectious clone, we were able to create a heat and pH stable virus that had delayed viral uncoating during the infectious lifecycle. Cryo-EM reconstructions of the triple substitution virus demonstrated that enhanced inter-chain hydrogen bonding was vital for increased capsid stability. Finally, mutagenesis to remove the enhanced inter-chain hydrogen bonding reverted capsid stability back to wild-type levels. This work contributes to fundamental calicivirus biology by demonstrating areas of importance in capsid stability down to amino acid resolution. Furthermore, this work could inform vaccine design for a thermostable norovirus vaccine in the future. Author SummaryNoroviruses cause gastroenteritis and globally contribute to the death of up to 250,000 people worldwide per annum and an estimated healthcare cost of $4 billion. Despite this, there is no vaccine or antiviral treatment available, thus more work needs to be conducted to understand the mechanisms that underpin the viral life cycle. The norovirus capsid is a meta-stable shell-like structure evolved to protect the viral RNA from the harsh external environment, until cellular triggers allow genome release to initiate infection of a host cell. However, the molecular interactions that are key for controlling this balance are relatively unstudied. In this report, we identify that hydrogen bonding at the capsid protomer-protomer interface are vital for maintaining this balance, with increased inter-molecular hydrogen bonding at three specific amino acids able to increase viral stability whilst still permitting infectious genome release. Furthermore, reducing inter-molecular hydrogen bonding at these crucial amino acids was able to reverse this mechanism. These results should inform future norovirus vaccine studies, where thermostable virus-like-particles are needed to overcome issues with cold-chain storage.

microbiology↗

bioRxiv2026

Inhibition of VP2-mediated entry: a potential antiviral strategy to treat or prevent calicivirus disease

The Caliciviridae include many notable human and animal pathogens, including norovirus and sapovirus, which cause outbreaks of acute gastroenteritis. We previously demonstrated that, following receptor engagement, feline calicivirus (FCV) assembles a portal structure at a unique capsid three-fold axis. This comprises twelve copies of the minor capsid protein VP2 and is essential for genome delivery. We designed a short peptide based on our structure data that occludes the VP2 binding sites on the capsid surface, to prevent assembly of the VP2 portal and thereby halt the viral entry mechanism. Incubation with low micromolar concentrations of the peptide considerably reduced the infectivity of two laboratory strains and four clinical isolates of FCV associated with respiratory or virulent-systemic disease. Cryo-electron microscopy structures of FCV virions complexed with the peptide confirmed that the peptide occupies the VP2 binding site on the major capsid protein VP1, preventing portal assembly and subsequent genome delivery. Our data show that targeting VP2 is a viable antiviral approach to preventing calicivirus infection, with potential for the treatment or prevention of norovirus disease.

microbiology↗

bioRxiv2025

Phosphorylation enables allosteric control of a viral condensate

In many viruses, intrinsically disordered proteins (IDPs) drive the formation of replicative organelles essential for viral production. In species A rotaviruses, the disordered protein NSP5 forms condensates in cells via liquid-liquid phase separation (LLPS). Yet the sequence diversity of NSP5 raises the question of whether condensate formation is conserved across all strains and if distinct variants employ alternative mechanisms for nucleating phase separation. Using a machine learning approach, we demonstrate that NSP5 variants differ significantly in their propensity to phase-separate. We engineered a variant incorporating amino acid signatures from strains with low LLPS tendency, which failed to phase separate in vitro yet supported the formation of replicative condensates in recombinant viruses in cells. Low-tendency LLPS strains require phosphorylation of NSP5 to nucleate phase separation, whereas high-tendency strains do not, suggesting distinct nucleation mechanisms. Furthermore, hydrogen-deuterium exchange mass spectrometry revealed a phosphorylation-driven allosteric switch between binding sites on the high-propensity variant. These findings establish that phosphorylation plays a context-dependent role in the formation of replicative organelles across diverse rotaviruses.

biophysics↗

bioRxiv2024

Upscaling production of immunogenic poliovirus virus-like particles in Pichia Pastoris by controlled fermentation

The success of the poliovirus (PV) vaccines has enabled the near-eradication of wild PV, however, their continued use post-eradication poses a significant threat to maintaining a polio-free world. Recombinant virus-like particles (VLPs) that lack the viral genome remove this risk. Here, we demonstrate the production of PV VLPs for all three serotypes by controlled fermentation using Pichia pastoris. We determined the cryo-EM structure of a new PV-2 mutant, termed SC5a, in comparison to PV-2 SC6b VLPs described previously and investigated the immunogenicity of PV-2 SC5a VLPs. Finally, a trivalent immunogenicity trial using bioreactor-derived VLPs of all three serotypes in the presence of Alhydrogel adjuvant, showed that these VLPs outperform the current IPV vaccine in the standard vaccine potency assay, offering the potential for dose-sparing. Overall, these results provide further evidence that yeast-produced VLPs have the potential to be a next-generation polio vaccine in a post-eradication world.

microbiology↗

bioRxiv2024

A VLP vaccine platform comprising the core protein of hepatitis B virus with N-terminal antigen capture

Nanoparticle presentation systems offer the potential to develop new vaccines rapidly in response to emerging diseases, a public health need that has become increasingly evident in the wake of the COVID-19 pandemic. Previously, we reported a nanoparticle scaffold system termed VelcroVax. This was constructed by insertion of a high affinity SUMO binding protein (Affimer), able to recognise a SUMO peptide tag, into the major immunodominant region of VLPs assembled from a tandem (fused dimer) form of hepatitis B virus (HBV) core protein (HBc). Here we describe a modified form of VelcroVax, comprising monomeric HBc with the Affimer inserted at the N-terminus (termed N-VelcroVax). In contrast to the tandem form of VelcroVax, N-VelcroVax VLPs were expressed well in E. coli. The VLPs effectively bound SUMO-tagged Junin virus glycoprotein, gp1 as assessed by structural and serological analyses. Cryo-EM characterisation of N-VelcroVax complexed with a SUMO-Junin gp1 showed continuous density attributable to the fused Affimer, in addition to evidence of target antigen capture. Collectively, these data suggest that N-VelcroVax has potential as a versatile next generation vaccine scaffold.

molecular biology↗

bioRxiv2024

Risk-free polio vaccine: Recombinant expression systems for production of stabilised virus-like particles

Polioviruses have caused crippling disease in humans for centuries, prior to the successful development of vaccines in the mid-1900s, which dramatically reduced disease prevalence. Continued use of these vaccines, however, threatens ultimate disease eradication and achievement of a polio-free world. Virus-like particles (VLPs) that lack a viral genome represent a safer potential vaccine, although they require particle stabilization. Using our previously established genetic techniques to stabilize the structural capsid proteins, we demonstrate production of poliovirus VLPs of all three serotypes, from four different recombinant expression systems. We compare the antigenicity, thermostability and immunogenicity of these stabilized VLPs against the current inactivated polio vaccine, demonstrating equivalent or superior immunogenicity. Structural analyses of these recombinant VLPs provide a rational understanding of the stabilizing mutations and the role of potential excipients. Collectively, we have established these poliovirus stabilized VLPs as viable next-generation vaccine candidates for the future.

microbiology↗

bioRxiv2022

First gene-edited calf with reduced susceptibility to a major viral pathogen

Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 16 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in CD46 may reduce the burden of BVDV-associated diseases in cattle, and is consistent with our stepwise, in vitro and ex vivo experiments with cell lines and matched fetal clones.

genomics↗

bioRxiv2022

Protease-independent production of poliovirus virus-like particles in Pichia pastoris: Implications for efficient vaccine development and insights into capsid assembly.

The production of enterovirus virus-like particles (VLPs) which lack the viral genome have great potential as vaccines for a number of diseases, such as poliomyelitis and hand, foot-and-mouth disease. These VLPs can mimic empty capsids, which are antigenically indistinguishable from mature virions, produced naturally during viral infection. Both in infection and in vitro, capsids and VLPs are generated by the cleavage of the P1 precursor protein by a viral protease. Here, using a stabilised poliovirus 1 (PV-1) P1 sequence as an exemplar, we show the production of PV-1 VLPs in Pichia pastoris in the absence of the potentially cytotoxic protease, 3CD, instead using the porcine teschovirus 2A (P2A) peptide sequence to terminate translation between individual capsid proteins. We compare this to protease-dependent production of PV-1 VLPs. Analysis of all permutations of the order of the capsid protein sequences revealed that only VP3 could be tagged with P2A and maintain native antigenicity. Transmission electron microscopy of these VLPs reveals the classic picornaviral icosahedral structure. Furthermore, these particles were thermostable above 37{degrees}C, demonstrating their potential as next generation vaccine candidates for PV. Finally, we believe the demonstration that native antigenic VLPs can be produced using protease-independent methods opens the possibility for future enteroviral vaccines to take advantage of recent vaccine technological advances, such as adenovirus-vectored vaccines and mRNA vaccines, circumventing the potential problems of cytotoxicity associated with 3CD, allowing for the production of immunogenic enterovirus VLPs in vivo.

microbiology↗

bioRxiv2022

Production and characterisation of stabilised PV-3 virus-like particles using Pichia pastoris

Following the success of global vaccination programmes using the live-attenuated oral and inactivated poliovirus vaccines (OPV and IPV), wild poliovirus (PV) is now only endemic in Afghanistan and Pakistan. However, the continued use of these vaccines poses potential risks to the eradication of PV. The production of recombinant PV virus-like particles (VLPs), which lack the viral genome offer great potential as next-generation vaccines for the post-polio world. We have previously reported production of PV VLPs using Pichia pastoris, however, these VLPs were in the non-native conformation (C Ag), which would not produce effective protection against PV. Here, we build on this work and show that it is possible to produce wt PV-3 and thermally-stabilised PV-3 (referred to as PV-3 SC8) VLPs in the native conformation (D Ag) using Pichia pastoris. We show that the PV-3 SC8 VLPs provide a much-improved D:C antigen ratio as compared to wt PV-3, whilst exhibiting greater thermostability than the current IPV vaccine. Finally, we determine the cryo-EM structure of the yeast-derived PV-3 SC8 VLPs and compare this to previously published PV-3 D Ag structures, highlighting the similarities between these recombinantly-expressed VLPs and the infectious virus, further emphasising their potential as a next-generation vaccine candidate for PV.

microbiology↗

bioRxiv2022

A conserved glutathione binding site in poliovirus is a target for antivirals and vaccine stabilisation

Strategies to prevent the recurrence of poliovirus (PV) after eradication may utilise non-infectious, recombinant virus-like particle (VLP) vaccines. Despite clear advantages over inactivated or attenuated virus vaccines, instability of VLPs can compromise their immunogenicity. Glutathione (GSH), an important cellular reducing agent, is a crucial co-factor for the morphogenesis of enteroviruses, including PV. We report cryo-EM structures of GSH bound to PV serotype 3 VLPs showing that it can enhance particle stability. GSH binds the positively charged pocket at the interprotomer interface shown recently to bind GSH in enterovirus F3 and putative antiviral benzene sulphonamide compounds in other enteroviruses. We show, using high-resolution cryo-EM, the binding of a benzene sulphonamide compound with a PV serotype 2 VLP, consistent with antiviral activity through over-stabilizing the interprotomer pocket, preventing the capsid rearrangements necessary for viral infection. Collectively, these results suggest GSH or an analogous tight-binding antiviral offers the potential for stabilizing VLP vaccines.

biochemistry↗

bioRxiv2022

VelcroVax: a bolt-on vaccine platform technology improves antibody titres against a viral glycoprotein in mice

Having varied approaches to the design and manufacture of vaccines is critical in being able to respond to worldwide needs and to newly emerging pathogens. Virus-like particle (VLP) vaccines form the basis of two of the most successful licensed vaccines (against hepatitis B virus (HBV) and human papillomavirus). They are produced by recombinant expression of viral structural proteins, which self-assemble into immunogenic nanoparticles. VLPs can also be modified to present unrelated antigens, and here we describe a universal bolt-on vaccine platform (termed VelcroVax) where the capturing VLP and the target antigen (hapten) are produced separately. We utilise a modified HBV core (HBcAg) VLP, with surface expression of a high-affinity binding sequence (Affimer) directed against a SUMO tag and use this to capture SUMO-tagged gp1 glycoprotein from the arenavirus, Junin virus (JUNV). Using this model system, we have solved high-resolution structures of VelcroVax VLPs, and shown that the VelcroVax-JUNV gp1 complex induces superior humoral immune responses compared to the non-complexed viral protein. We propose that this system could be modified to present a range of haptens and therefore form the foundation of future rapid-response vaccination strategies.

immunology↗

bioRxiv2021

Recurrent Vulvovaginal Candidiasis; a dynamic interkingdom biofilm disease of Candida and Lactobacillus

Vulvovaginal Candidiasis (VVC) is the most prevalent Candida infection in humans affecting 75% of women at least once throughout their lifetime. In its debilitating recurrent form, RVVC is estimated to affect 140 million women annually. Despite this strikingly high prevalence, treatment options for RVVC remain limited with many women experiencing failed clinical treatment with frontline azoles. Further, the cause of onset and recurrence of disease is largely unknown with few studies identifying potential mechanisms of failed treatment. This study aimed to assess a panel of clinical samples from healthy women and those with RVVC to investigate the influence of Candida, vaginal microbiome and antagonism between Candida and Lactobacillus on disease pathology. 16S rRNA sequencing characterised disease by a reduction in specific health-associated Lactobacillus such as L. crispatus, coupled with an increase in L. iners. In vitro analysis showed Candida albicans clinical isolates are capable of heterogeneous biofilm formation and show the presence of hyphae and C. albicans aggregates in vaginal lavage. Additionally, the ability of Lactobacillus to inhibit C. albicans biofilm formation and biofilm-related gene expression was demonstrated. Using RNA sequencing technology, we were able to exploit a possible mechanism by which L. crispatus may aim to re-establish a healthy vaginal environment through amino-acid acquisition from C. albicans. This study suggests RVVC is not entirely due to an arbitrary switch in C. albicans from commensal to pathogen and understanding interactions between the yeast and vaginal Lactobacillus species may be more crucial to elucidating the cause of RVVC and developing appropriate therapies.

microbiology↗

bioRxiv2021

Structural insight into Pichia pastoris fatty acid synthase

Type I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products. Serendipitously, we identified FAS as a contaminant in a cryoEM dataset of virus-like particles (VLPs) purified from P. pastoris, an important model organism and common expression system used in protein production. From these data, we determined the structure of P. pastoris FAS to 3.1 [A] resolution. While the overall organisation of the complex was typical of type I FASs, we identified several differences in both structural and enzymatic domains through comparison with the prototypical yeast FAS from S. cerevisiae. Using focussed classification, we were also able to resolve and model the mobile acyl-carrier protein (ACP) domain, which is key for function. Ultimately, the structure reported here will be a useful resource for further efforts to engineer yeast FAS for synthesis of alternate products.

microbiology↗

bioRxiv2020

Adaptation to host cell environment during experimental evolution of Zika virus

Zika virus (ZIKV) infection can cause important developmental and neurological defects in Humans. Type I/III interferon responses control ZIKV infection and pathological processes, yet the virus has evolved various mechanisms to defeat these host responses. Here, we established a pipeline to delineate at high-resolution the genetic evolution of ZIKV in a controlled host cell environment. We uncovered that serially passaged ZIKV acquired increased infectivity and simultaneously developed a resistance to TLR3-induced restriction. We built a mathematical model that suggests that the increased infectivity is due to a reduced time-lag between infection and viral replication. We found that this adaptation is cell-type specific, suggesting that different cell environments may drive viral evolution along different routes. Deep-sequencing of ZIKV populations pinpointed mutations whose increased frequencies temporally coincide with the acquisition of the adapted phenotype. We functionally validated S455L, a substitution in ZIKV envelope (E) protein, recapitulating the adapted phenotype. Its positioning on the E structure suggests a putative function in protein refolding/stability. Taken together, our results uncovered ZIKV adaptations to the cellular environment leading to accelerated replication onset coupled with resistance to TLR3-induced antiviral response. Our work provides insights into Zika virus adaptation to host cells and immune escape mechanisms.

microbiology↗

bioRxiv2018

Antiviral Response by Plasmacytoid Dendritic Cells via Interferogenic Synapse with Infected Cells

Type I interferon (IFN-I) is critical for protection against viral infections. Plasmacytoid dendritic cells (pDCs) massively produce IFN-I against viruses. Physical contacts are required for pDC-mediated sensing of cells infected by genetically distant viruses. How and why these contacts are established remains enigmatic. Using dengue, hepatitis C, zika viruses, we demonstrate that the pDC/infected cell interface is a specialized platform for viral immunostimulatory-RNA transfer, which we named interferogenic synapse and required for pDC-mediated antiviral response. This synapse is an exquisitely differentiated territory with polarized adhesion complexes and regulators of actin network and endocytosis. Toll-like receptor 7-induced signaling in pDCs promotes the interferogenic synapse establishment, thus providing a feed-forward regulation that sustains contacts with infected cells. We propose that the interferogenic synapse is crucial to pDC function as it allows scanning of infected cells to locally secrete IFN-I at the infection site, thereby confining a response potentially deleterious to the host.\n\nHighlightsO_LIpDCs adhere to infected cells via L{beta}2 integrin/ICAM-1\nC_LIO_LIRegulators of actin network and endocytosis polarize at contact\nC_LIO_LITLR7-induced signaling potentiates pDC polarity\nC_LIO_LIInfected cells activate pDCs by interferogenic synapse\nC_LI

microbiology↗