Krios I-Titan Krios I at Diamond
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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.
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Mar 2025
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Krios I-Titan Krios I at Diamond
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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.
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Jan 2025
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Sabine
Bou-Antoun
,
Sakib
Rokadiya
,
Diane
Ashiru-Oredope
,
Alicia
Demirjian
,
Emma
Sherwood
,
Nicholas
Ellaby
,
Sarah
Gerver
,
Carlota
Grossi
,
Katie
Harman
,
Hassan
Hartman
,
Alessandra
Lochen
,
Manon
Ragonnet-Cronin
,
Hanna
Squire
,
J. Mark
Sutton
,
Simon
Thelwall
,
Julia
Tree
,
Mohammad W.
Bahar
,
David I.
Stuart
,
Colin S
Brown
,
Meera
Chand
,
Susan
Hopkins
Open Access
Abstract: The COVID-19 pandemic saw unprecedented resources and funds driven into research for the development, and subsequent rapid distribution, of vaccines, diagnostics and directly acting antivirals (DAAs). DAAs have undeniably prevented progression and life-threatening conditions in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, there are concerns of antimicrobial resistance (AMR), antiviral resistance specifically, for DAAs. To preserve activity of DAAs for COVID-19 therapy, as well as detect possible mutations conferring resistance, antimicrobial stewardship and surveillance were rapidly implemented in England. This paper expands on the ubiquitous ongoing public health activities carried out in England, including epidemiologic, virologic and genomic surveillance, to support the stewardship of DAAs and assess the deployment, safety, effectiveness and resistance potential of these novel and repurposed therapeutics.
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Nov 2023
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Krios IV-Titan Krios IV at Diamond
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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.
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Nov 2022
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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.
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Oct 2022
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I03-Macromolecular Crystallography
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Wanwisa
Dejnirattisai
,
Jiandong
Huo
,
Daming
Zhou
,
Jiří
Zahradník
,
Piyada
Supasa
,
Chang
Liu
,
Helen M. E.
Duyvesteyn
,
Helen M.
Ginn
,
Alexander J.
Mentzer
,
Aekkachai
Tuekprakhon
,
Rungtiwa
Nutalai
,
Beibei
Wang
,
Aiste
Dijokaite
,
Suman
Khan
,
Ori
Avinoam
,
Mohammad
Bahar
,
Donal
Skelly
,
Sandra
Adele
,
Sile Ann
Johnson
,
Ali
Amini
,
Thomas
Ritter
,
Chris
Mason
,
Christina
Dold
,
Daniel
Pan
,
Sara
Assadi
,
Adam
Bellass
,
Nikki
Omo-Dare
,
David
Koeckerling
,
Amy
Flaxman
,
Daniel
Jenkin
,
Parvinder K.
Aley
,
Merryn
Voysey
,
Sue Ann
Costa Clemens
,
Felipe Gomes
Naveca
,
Valdinete
Nascimento
,
Fernanda
Nascimento
,
Cristiano
Fernandes Da Costa
,
Paola Cristina
Resende
,
Alex
Pauvolid-Correa
,
Marilda M.
Siqueira
,
Vicky
Baillie
,
Natali
Serafin
,
Gaurav
Kwatra
,
Kelly
Da Silva
,
Shabir A.
Madhi
,
Marta C.
Nunes
,
Tariq
Malik
,
Peter J. M.
Openshaw
,
J. Kenneth
Baillie
,
Malcolm G.
Semple
,
Alain R.
Townsend
,
Kuan-Ying A.
Huang
,
Tiong Kit
Tan
,
Miles W.
Carroll
,
Paul
Klenerman
,
Eleanor
Barnes
,
Susanna J.
Dunachie
,
Bede
Constantinides
,
Hermione
Webster
,
Derrick
Crook
,
Andrew J.
Pollard
,
Teresa
Lambe
,
Neil G.
Paterson
,
Mark A.
Williams
,
David R.
Hall
,
Elizabeth E.
Fry
,
Juthathip
Mongkolsapaya
,
Jingshan
Ren
,
Gideon
Schreiber
,
David I.
Stuart
,
Gavin R.
Screaton
Diamond Proposal Number(s):
[27009]
Open Access
Abstract: On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate Omicron-B.1.1.529 was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses.
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Jan 2022
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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.
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Jan 2021
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Poliovirus (PV) is the causative agent of poliomyelitis, a crippling human disease known since antiquity. PV occurs in two distinct antigenic forms, D and C, of which only the D form elicits a robust neutralizing response. Developing a synthetically produced stabilized virus-like particle (sVLP)-based vaccine with D antigenicity, without the drawbacks of current vaccines, will be a major step towards the final eradication of poliovirus. Such a sVLP would retain the native antigenic conformation and the repetitive structure of the original virus particle, but lack infectious genomic material. In this study, we report the production of synthetically stabilized PV VLPs in plants. Mice carrying the gene for the human PV receptor are protected from wild-type PV when immunized with the plant-made PV sVLPs. Structural analysis of the stabilized mutant at 3.6 Å resolution by cryo-electron microscopy and single-particle reconstruction reveals a structure almost indistinguishable from wild-type PV3.
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Aug 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8423]
Abstract: Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae, infects young salmon, with a severe impact on the commercial sea farming industry. Of the five mature proteins encoded by the IPNV genome, the multifunctional VP3 has an essential role in morphogenesis; interacting with the capsid protein VP2, the viral double-stranded RNA (dsRNA) genome and the RNA-dependent RNA polymerase VP1. Here we investigate one of these VP3 functions and present the crystal structure of the C-terminal 12 residues of VP3 bound to the VP1 polymerase. This interaction, visualized for the first time, reveals the precise molecular determinants used by VP3 to bind the polymerase. Competition binding studies confirm that this region of VP3 is necessary and sufficient for VP1 binding, while biochemical experiments show that VP3 attachment has no effect on polymerase activity. These results indicate how VP3 recruits the polymerase into birnavirus capsids during morphogenesis.
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Feb 2013
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: The RNA-dependent RNA polymerase VP1 of infectious pancreatic necrosis virus (IPNV) is a single polypeptide responsible for both viral RNA transcription and genome replication. Sequence analysis identifies IPNV VP1 as having an unusual active site topology. We have purified, crystallized and solved the structure of IPNV VP1 to 2.3 Å resolution in its apo form and at 2.2 Å resolution bound to the catalytically-activating metal magnesium. We find that recombinantly-expressed VP1 is highly active for RNA transcription and replication, yielding both free and polymerase-attached RNA products. IPNV VP1 also possesses terminal (deoxy)nucleotide transferase, RNA-dependent DNA polymerase (reverse transcriptase) and template-independent self-guanylylation activity. The N-terminus of VP1 interacts with the active-site cleft and we show that the N-terminal serine residue is required for formation of covalent RNA:polymerase complexes, providing a mechanism for the genesis of viral genome:polymerase complexes observed in vivo.
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Dec 2011
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