I03-Macromolecular Crystallography
|
Marie
Bonnet-Di Placido
,
Helen M. E.
Duyvesteyn
,
Angela W.
Steyn
,
Abigail L.
Hay
,
Claudine
Porta
,
Kristel Ramirez
Valdez
,
Elena
Lokhman
,
Sylvia
Crossley
,
Kevan
Hanson
,
William N.
Mwangi
,
Danish
Munir
,
Eva
Perez-Martin
,
Nick J.
Knowles
,
Alison
Burman
,
Abdelaziz A.
Yassin
,
Amin
Asfor
,
Cristina
Faralla
,
Katherine J.
Lam
,
Róisín
Mccomb
,
Carina
Leifeld
,
Kimberly
Pietersz
,
Donald P.
King
,
Erwin
Van Den Born
,
Sherie K.
Duncan
,
Bryan
Charleston
,
Elizabeth E.
Fry
,
Jingshan
Ren
,
David I.
Stuart
,
John A.
Hammond
Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Foot-and-mouth disease virus (FMDV) causes a devastating disease that threatens global food security. Vaccination is hindered by antigenic diversity across serotypes. To identify cross-serotype neutralising epitopes, we isolated 24 FMDV-specific antibodies from cattle sequentially vaccinated with antigens from four serotypes, of which three neutralised three vaccine strains. These three antibodies neutralised 21 and bound 59 additional topotypes across O, A, Asia 1, and C serotypes. Cryo-EM complexes of Fabs with FMD virus-like particles indicated all three recognise a common flexible epitope at the VP1 C-terminus, confirmed by binding competition. Crystallography and structural modelling revealed that a normally inaccessible surface of the hydrophobic VP1 C-terminal peptides inserts into a similar groove in all three antibodies. Comparison of neutralisation activity and integrin receptor blocking by whole antibodies, F(ab’)2s, and Fabs suggests neutralisation is mediated by Fc steric hindrance of receptor binding. This cryptic, linear, and cross-serotype neutralising epitope may inform improved FMD vaccines.
|
Apr 2026
|
|
Krios I-Titan Krios I at Diamond
|
Lee
Sherry
,
Keith
Grehan
,
Mohammad W.
Bahar
,
Jessica J.
Swanson
,
Helen
Fox
,
Sue
Matthews
,
Sarah
Carlyle
,
Ling
Qin
,
Claudine
Porta
,
Steven
Wilkinson
,
Suzanne
Robb
,
Naomi
Clark
,
John
Liddell
,
Elizabeth E.
Fry
,
David I.
Stuart
,
Andrew J.
Macadam
,
David J.
Rowlands
,
Nicola J.
Stonehouse
Diamond Proposal Number(s):
[28713]
Open Access
Abstract: The success of the poliovirus (PV) vaccines has enabled the near-eradication of wild PV, however, their continued use post-eradication poses concerns, due to the potential for virus escape during vaccine manufacture. 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 PV2 mutant, termed SC5a, in comparison to PV2-SC6b VLPs described previously and investigated the immunogenicity of PV2-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.
|
Mar 2025
|
|
Krios I-Titan Krios I at Diamond
|
Lee
Sherry
,
Mohammad W.
Bahar
,
Claudine
Porta
,
Helen
Fox
,
Keith
Grehan
,
Veronica
Nasta
,
Helen M. E.
Duyvesteyn
,
Luigi
De Colibus
,
Johanna
Marsian
,
Inga
Murdoch
,
Daniel
Ponndorf
,
Seong-Ryong
Kim
,
Sachin
Shah
,
Sarah
Carlyle
,
Jessica J.
Swanson
,
Sue
Matthews
,
Clare
Nicol
,
George P.
Lomonossoff
,
Andrew J.
Macadam
,
Elizabeth E.
Fry
,
David I.
Stuart
,
Nicola J.
Stonehouse
,
David J.
Rowlands
Diamond Proposal Number(s):
[14856, 20223]
Open Access
Abstract: Polioviruses have caused crippling disease in humans for centuries, prior to the successful development of vaccines in the mid-1900’s, 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 in female Wistar rats. 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.
|
Jan 2025
|
|
|
|
John D.
Clarke
,
Helen M. E.
Duyvesteyn
,
Eva
Perez-Martin
,
Undīne
Latišenko
,
Claudine
Porta
,
Kathleen V.
Humphreys
,
Abigail L.
Hay
,
Jingshan
Ren
,
Elizabeth E.
Fry
,
Erwin
Van Den Born
,
Bryan
Charleston
,
Marie
Bonnet-Di Placido
,
Raymond J.
Owens
,
David I.
Stuart
,
John A.
Hammond
Open Access
Abstract: Foot-and-mouth disease vaccination using inactivated virus is suboptimal, as the icosahedral viral capsids often disassemble into antigenically distinct pentameric units during long-term storage, or exposure to elevated temperature or lowered pH, and thus raise a response that is no longer protective. Furthermore, as foot-and-mouth disease virus (FMDV)’s seven serotypes are antigenically diverse, cross-protection from a single serotype vaccine is limited, and most existing mouse and bovine antibodies and camelid single-domain heavy chain-only antibodies are serotype-specific. For quality control purposes, there is a real need for pan-serotype antibodies that clearly distinguish between pentamer (12S) and protective intact FMDV capsid. To date, few cross-serotype bovine-derived antibodies have been reported in the literature. We identify a bovine antibody with an ultralong CDR-H3, Ab117, whose structural analysis reveals that it binds to a deep, hydrophobic pocket on the interior surface of the capsid via the CDR-H3. Main-chain and hydrophobic interactions provide broad serotype specificity. ELISA analysis confirms that Ab117 is a novel pan-serotype and conformational epitope-specific 12S reagent, suitable for assessing capsid integrity.
|
Oct 2024
|
|
Krios IV-Titan Krios IV at Diamond
|
Mohammad W.
Bahar
,
Veronica
Nasta
,
Helen
Fox
,
Lee
Sherry
,
Keith
Grehan
,
Claudine
Porta
,
Andrew J.
Macadam
,
Nicola J.
Stonehouse
,
David J.
Rowlands
,
Elizabeth E.
Fry
,
David I.
Stuart
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: 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.
|
Nov 2022
|
|
|
|
Open Access
Abstract: 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.
|
Oct 2022
|
|
|
|
Open Access
Abstract: Global vaccination programs using live-attenuated oral and inactivated polio vaccine (OPV and IPV) have almost eradicated poliovirus (PV) but these vaccines or their production pose significant risk in a polio-free world. Recombinant PV virus-like particles (VLPs), lacking the viral genome, represent safe next-generation vaccines, however their production requires optimisation. Here we present an efficient mammalian expression strategy producing good yields of wild-type PV VLPs for all three serotypes and a thermostabilised variant for PV3. Whilst the wild-type VLPs were predominantly in the non-native C-antigenic form, the thermostabilised PV3 VLPs adopted the native D-antigenic conformation eliciting neutralising antibody titres equivalent to the current IPV and were indistinguishable from natural empty particles by cryo-electron microscopy with a similar stabilising lipidic pocket-factor in the VP1 β-barrel. This factor may not be available in alternative expression systems, which may require synthetic pocket-binding factors. VLPs equivalent to these mammalian expressed thermostabilized particles, represent safer non-infectious vaccine candidates for the post-eradication era.
|
Jan 2021
|
|
M01-Polara at OPIC (Oxford)
|
Ling
Zhu
,
Xiangxi
Wang
,
Jingshan
Ren
,
Claudine
Porta
,
Hannah
Wenham
,
Jens-Ola
Ekström
,
Anusha
Panjwani
,
Nick J.
Knowles
,
Abhay
Kotecha
,
C. Alistair
Siebert
,
A. Michael
Lindberg
,
Elizabeth E.
Fry
,
Zihe
Rao
,
Tobias J.
Tuthill
,
David I.
Stuart
Open Access
Abstract: Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses.
|
Dec 2015
|
|
I24-Microfocus Macromolecular Crystallography
|
Abhay
Kotecha
,
Julian
Seago
,
Katherine
Scott
,
Alison
Burman
,
Silvia
Loureiro
,
Jingshan
Ren
,
Claudine
Porta
,
Helen M
Ginn
,
Terry
Jackson
,
Eva
Perez-Martin
,
C Alistair
Siebert
,
Guntram
Paul
,
Juha T
Huiskonen
,
Ian M
Jones
,
Robert
Esnouf
,
Elizabeth
Fry
,
Francois F
Maree
,
Bryan
Charleston
,
Dave
Stuart
Diamond Proposal Number(s):
[10627]
Abstract: Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.
|
Sep 2015
|
|
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Jingshan
Ren
,
Xiangxi
Wang
,
Zhongyu
Hu
,
Qiang
Gao
,
Yao
Sun
,
Xuemei
Li
,
Claudine
Porta
,
Thomas
Walter
,
Robert
Gilbert
,
Yuguang
Zhao
,
Danny
Axford
,
Mark
Williams
,
Katherine
Mcauley
,
David J.
Rowlands
,
Weidong
Yin
,
Junzhi
Wang
,
David I.
Stuart
,
Zihe
Rao
,
Elizabeth E.
Fry
Open Access
Abstract: It remains largely mysterious how the genomes of non-enveloped eukaryotic viruses are transferred across a membrane into the host cell. Picornaviruses are simple models for such viruses, and initiate this uncoating process through particle expansion, which reveals channels through which internal capsid proteins and the viral genome presumably exit the particle, although this has not been clearly seen until now. Here we present the atomic structure of an uncoating intermediate for the major human picornavirus pathogen CAV16, which reveals VP1 partly extruded from the capsid, poised to embed in the host membrane. Together with previous low-resolution results, we are able to propose a detailed hypothesis for the ordered egress of the internal proteins, using two distinct sets of channels through the capsid, and suggest a structural link to the condensed RNA within the particle, which may be involved in triggering RNA release.
|
Jun 2013
|
|
