Biology subjects

Snowden, J. S.

Publications and source records attributed to Snowden, J. S..

7 recordsLinked to original sources

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

Mechanism of enterovirus VP0 maturation cleavage based on the structure of a stabilised assembly intermediate.

Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0. We have demonstrated an essential role of VP0 E096 in VP0 cleavage independent of RNA encapsidation and generated a new model of capsid maturation, supported by bioinformatic analysis. This provides a molecular basis for RNA-dependence, where RNA induces conformational changes required for VP0 maturation, but that RNA packaging itself is not sufficient to induce maturation. These data have implications for understanding production of infectious virions and potential relevance for future vaccine and antiviral drug design.

microbiology↗

bioRxiv2023

Amino acid substitutions in norovirus VP1 dictate cell tropism via an attachment process dependent on membrane mobility.

Viruses interact with receptors on the cell surface to initiate and co-ordinate infection. The distribution of receptors on host cells can be a key determinant of viral tropism and host infection. Unravelling the complex nature of virus-receptor interactions is, therefore, of fundamental importance to understanding viral pathogenesis. Noroviruses are non-enveloped, icosahedral, positive-sense RNA viruses of global importance to human health, with no approved vaccine or antiviral agent available. Here we use murine norovirus as a model for the study of molecular mechanisms of virus-receptor interactions. We show that variation at a single amino acid residue in the major viral capsid protein had a key impact on the interaction between virus and receptor. This variation did not affect virion production or virus growth kinetics, but a specific amino acid was rapidly selected through evolution experiments, and significantly improved cellular attachment when infecting immune cells in suspension. However, reducing plasma membrane mobility counteracted this phenotype, providing insight into for the role of membrane fluidity and receptor recruitment in norovirus cellular attachment. When the infectivity of a panel of recombinant viruses with single amino acid variations was compared in vivo, there were significant differences in the distribution of viruses in a murine model, demonstrating a role in cellular tropism in vivo. Overall, these results highlight the importance of lipid rafts and virus-induced receptor recruitment in viral infection, as well as how capsid evolution can greatly influence cellular tropism, within-host spread and pathogenicity. ImportanceAll viruses initiate infection by utilising receptors to attach to target host cells. These virus-receptor interactions can therefore dictate viral replication and pathogenesis. Understanding the nature of virus-receptor interactions could also be important to developing novel therapies. Noroviruses are non-enveloped icosahedral viruses of medical importance. They are a common cause of acute gastroenteritis with no approved vaccine or therapy and are a tractable model for studying fundamental virus biology. In this study, we utilise the murine norovirus model system to show that variation in a single amino acid of the major capsid protein can alone can affect viral infectivity through improved attachment to suspension cells. Reducing plasma membrane mobility reduced infectivity, providing an insight into the importance of membrane mobility for receptor recruitment. Furthermore, variation at this site was able to change viral distribution in a murine model, illustrating how in-host capsid evolution can influence viral infectivity and immune evasion.

microbiology↗

bioRxiv2023

Production of antigenically stable enterovirus A71 virus-like particles in Pichia pastoris as a vaccine candidate.

Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are produced during the viral lifecycle. While initially antigenically indistinguishable from virions, ECs readily convert to an expanded conformation at moderate temperatures. In the closely related poliovirus, these conformational changes result in loss of antigenic sites required to elicit protective immune responses. Whether this is true for EVA71 remains to be determined and is the subject of this investigation. We previously reported the selection of a thermally resistant EVA71 genogroup B2 population using successive rounds of heating and passage. The mutations found in the structural protein-coding region of the selected population conferred increased thermal stability to both virions and naturally produced ECs. Here, we introduced these mutations into a recombinant expression system to produce stabilised virus-like particles (VLPs) in Pichia pastoris. The stabilised VLPs retain the native virion-like antigenic conformation as determined by reactivity with a specific antibody. Structural studies suggest multiple potential mechanisms of antigenic stabilisation, however, unlike poliovirus, both native and expanded EVA71 particles elicited antibodies able to directly neutralise virus in vitro. Therefore, the anti-EVA71 neutralising antibodies are elicited by sites which are not canonically associated with the native conformation, but whether antigenic sites specific to the native conformation provide additional protective responses in vivo remains unclear. VLPs are likely to provide cheaper and safer alternatives for vaccine production and these data show that VLP vaccines are comparable with inactivated virus vaccines at inducing neutralising antibodies.

immunology↗

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

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↗

bioRxiv2019

High-Resolution Cryo-EM Reveals Dynamics in the Murine Norovirus Capsid

Rather than acting as rigid symmetrical shells to protect and transmit their genomes, the capsids of non-enveloped, icosahedral viruses co-ordinate multiple, essential processes during the viral life-cycle, and undergo extensive conformational rearrangements to deliver these functions. Capturing conformational flexibility has been challenging, yet could be key in understanding and combating infections that viruses cause. Noroviruses are non-enveloped, icosahedral viruses of global importance to human health. They are a common cause of acute non-bacterial gastroenteritis, yet no vaccines or antiviral agents specific to norovirus are available. Here, we use cryo-electron microscopy to study the high-resolution solution structures of infectious, inactivated and mutant virions of murine norovirus (MNV) as a model for human noroviruses. Together with genetic studies, we show that the viral capsid is highly dynamic. While there is little change to the shell domain of the capsid, the protruding domains that radiate from this are flexible and adopt distinct states both independently and synchronously. In doing so the viral capsid is able to sample a defined range of conformational space, with implications for the maintenance of virion stability and infectivity. These data will aid in developing the first generation of effective control measures against this virus.

microbiology↗