Biology subjects

Grehan, K.

Publications and source records attributed to Grehan, K..

9 recordsLinked to original sources

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

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↗

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

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

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↗

bioRxiv2018

The bat influenza H17N10 can be neutralized by broadly-neutralizing monoclonal antibodies and its neuraminidase can facilitate viral egress.

The diversity of subtypes within the Influenza A virus genus has recently expanded with the identification of H17N10 and H18N11 from bats. In order to further study the tropism and zoonotic potential of these viruses, we have successfully produced lentiviral pseudotypes bearing both haemagglutinin H17 and neuraminidase N10. These pseudotypes were shown to be efficiently neutralized by the broadly-neutralizing monoclonal antibodies CR9114 and FI6. Our studies also confirm previous reports that H17 does not use sialic acid as its cellular receptor, as pseudotypes bearing the H17 envelope glycoprotein are released into the cell supernatant in the absence of NA. However, we demonstrate that N10 facilitates heterosubtypic (H5 and H7) influenza HA-bearing pseudotype release in the absence of another source of NA, significantly increasing luciferase pseudotype production titres. Despite this, N10 shows no activity in the enzyme-linked lectin assay used for traditional sialidases. These findings suggest that this protein plays an important role in viral egress, but is perhaps involved in further accessory roles in the bat influenza lifecycle that are yet to be discovered. Thus we show the lentiviral pseudotype system is a useful research tool, and amenable for investigation of bat influenza tropism, restriction and sero-epidemiology, without the constraints or safety issues with producing a replication-competent virus, to which the human population is naive.\n\nSignificance statementInfluenza virus is responsible for mortality and morbidity across the globe; human populations are constantly at risk of newly emerging strains from the aquatic bird reservoir which harbors most of the subtypes of influenza A (H1-H16). Recently identified subtypes (H17N10, H18N11) from bats have broadened the reservoir from which potential pandemic strains of influenza can emerge. To evaluate the potential for these novel subtypes to cross over into human populations, their ability to establish an infection, in addition to the extent of cross-reactive immunity established by human seasonal strains needs to be investigated. This study highlights a novel platform for the study of the bat H17 and N10 envelope glycoproteins, using a lentiviral pseudotype system. Following the generation of this pseudotype it was employed in cell entry and microneutralization assays. These showed that two well-characterised monoclonal antibodies (mAb) which target avian and human influenza subtypes will also neutralize H17. Furthermore the data presented in this study show a novel aspect of the N10 glycoprotein in its ability to facilitate the budding of pseudotypes bearing different influenza HAs.

microbiology↗