I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946, 28534]
Open Access
Abstract: The spillover of New World (NW) arenaviruses from rodent reservoirs into human populations poses a continued risk to human health. NW arenaviruses present a glycoprotein (GP) complex on the envelope surface of the virion, which orchestrates host cell entry and is a key target of the immune response arising from infection and immunization. Each protomer of the trimeric GP is composed of a stable signal peptide, a GP1 attachment glycoprotein, and a GP2 fusion glycoprotein. To glean insights into the architecture of this key therapeutic target, we determined the crystal structures of NW GP1−GP2 heterodimeric complexes from Junín virus and Machupo virus. Due to the metastability of the interaction between GP1 and GP2, structural elucidation required the introduction of a disulfide bond at the GP1−GP2 complex interface, but no other stabilizing modifications were required. While the overall assembly of NW GP1−GP2 is conserved with that presented by Old World (OW) arenaviruses, including Lassa virus and lymphocytic choriomeningitis virus, NW GP1−GP2 complexes are structurally distinct. Indeed, we note that when compared to the OW GP1−GP2 complex, the globular portion of NW GP1 undergoes limited structural alterations upon detachment from its cognate GP2. We further demonstrate that our engineered GP1−GP2 heterodimers are antigenically relevant and recognized by neutralizing antibodies. These data provide insights into the distinct assemblies presented by NW and OW arenaviruses, as well as provide molecular-level blueprints that may guide vaccine development.
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Jun 2025
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Yanjun
Zhang
,
Yan
Wu
,
Meng-Qian
Zhang
,
Haiyue
Rao
,
Zhaoyong
Zhang
,
Xiangyue
He
,
Yiwen
Liang
,
Raoqing
Guo
,
Yaochang
Yuan
,
Jing
Sun
,
Helen M. E.
Duyvesteyn
,
Elizabeth E.
Fry
,
David I.
Stuart
,
Jingxian
Zhao
,
Xiaoyan
Pan
,
Shu-Lin
Liu
,
Jincun
Zhao
,
Jiandong
Huo
Open Access
Abstract: Current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are effective against severe disease and death, but do not prevent viral infections, probably due to the limited mucosal immunity induced by intramuscular administration of the vaccine. Fusion of SARS-CoV-2 subunit immunogens with a human IgG Fc backbone can be used as a mucosal vaccine but its effectiveness in delivery in animal models, and its immunogenicity and the vaccine-induced protection against viral infections requires further studies. Here we investigate a bivalent RBD-Fc vaccine that includes the spike receptor-binding domains (RBDs) of the ancestral and BQ.1.1 variant of SARS-CoV-2. Ex vivo fluorescent imaging demonstrates that this vaccine can be effectively delivered to the lungs of mice through intranasal administration, with enhancement of retention in the nasal cavity and lung parenchyma. In mice, the vaccine elicited potent and broad-spectrum antibody responses against different variants including KP.3 which could persist for at least 3 months after booster. Importantly, it was able to induce RBD-specific mucosal IgA responses. Further, heterologous intranasal immunisation with adeno-vectored Chadv1 and RBD-Fc elicited both potent neutralising antibody and T cell responses. Immunised BALB/c and K18-hACE2-transgenic mice were also protected against viral challenge of XBB.1 and viral transmission was effectively limited in hamsters through intranasal immunisation. This work thus demonstrates the potential of RBD-Fc antigens as mucosal vaccines for prevention of breakthrough infections and onward transmission. Moreover, Fc-fusion proteins can be used as an effective mucosal vaccine strategy which can be used either alone or in combination with other vaccine technology to constitute heterologous immunisations, enabling strong protection against SARS-CoV-2 and other respiratory viruses.
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May 2025
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I03-Macromolecular Crystallography
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Kalvis
Brangulis
,
Jill
Malfetano
,
Ashley L.
Marcinkiewicz
,
Alan
Wang
,
Yi-Lin
Chen
,
Jungsoon
Lee
,
Zhuyun
Liu
,
Xiuli
Yang
,
Ulrich
Strych
,
Dagnija
Tupina
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Inara
Akopjana
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Maria-Elena
Bottazzi
,
Utpal
Pal
,
Ching-Lin
Hsieh
,
Wen-Hsiang
Chen
,
Yi-Pin
Lin
Diamond Proposal Number(s):
[35587]
Open Access
Abstract: Borrelia burgdorferi (Bb) causes Lyme disease (LD), one of the most common vector-borne diseases in the Northern Hemisphere. Here, we solve the crystal structure of a mutated Bb vaccine antigen, CspZ-YA that lacks the ability to bind to host complement factor H (FH). We generate point mutants of CspZ-YA and identify CspZ-YAI183Y and CspZ-YAC187S to trigger more robust bactericidal responses. Compared to CspZ-YA, these CspZ-YA mutants require a lower immunization frequency to protect mice from LD-associated inflammation and bacterial colonization. Antigenicity of wild-type and mutant CspZ-YA proteins are similar, as measured using sera from infected people or immunized female mice. Structural comparison of CspZ-YA with CspZ-YAI183Y and CspZ-YAC187S shows enhanced interactions of two helices adjacent to the FH-binding sites in the mutants, consistent with their elevated thermostability. In line with these findings, protective CspZ-YA monoclonal antibodies show increased binding to CspZ-YA at a physiological temperature (37 °C). In summary, this proof-of-concept study applies structural vaccinology to enhance intramolecular interactions for the long-term stability of a Bb antigen while maintaining its protective epitopes, thus promoting LD vaccine development.
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Apr 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[12321, 34342]
Open Access
Abstract: Toll-like receptor (TLR) agonists are of interest in immunotherapy and cancer vaccines. The most common agonists of TLR2 are based on Pam2Cys or Pam3Cys. In the former, two palmitoyl (Pam) fatty acids are linked to a glycerylcysteine motif by ester linkages. Pam3Cys is analogous but contains an extra Pam group on the α-amine. Here, we compare the self-assembly in aqueous solution of the parent Pam2CysOH and Pam3Cys amino acid conjugates to that of Pam2CysSK4 and Pam3CysSK4 which are potent TLR2 agonists bearing the CysSK4 peptide sequence. All four conjugates exhibit a critical aggregation concentration above which self-assembled structures are formed. We find through a combination of small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and confocal fluorescence microscopy remarkable differences in self-assembled nanostructures. Pam2CysOH and Pam3CysOH both form unilamellar vesicles, although these are larger for the latter compound, an effect ascribed to enhanced membrane rigidity. This is in contrast to previously reported morphologies for Pam2CysSK4 and Pam3CysSK4, which are spherical micelles or predominantly wormlike micelles, respectively [Hamley, I. W.; et al. Toll-like Receptor Agonist Lipopeptides Self-Assemble into Distinct Nanostructures. Chem. Comm. 2014, 50, 15948-15951]. We also examine the effect of introduction in the bulky N-terminal Fmoc [fluorenylmethoxycarbonyl] group on the self-assembly of Fmoc-Pam2CysOH. This compound also forms vesicles (above a critical aggregation concentration, determined from dye probe fluorescence experiments) in aqueous solution, larger than those for Pam2CysOH and with a population of perforated/compound vesicles. The carboxyl-coated (and amino-coated for Pam2CysOH) vesicles demonstrated here represent a promising system for future development toward bionanotechnology applications such as immune therapies. Conjugates Pam2CysOH, Pam2CysSK4, and Pam3CysSK4 show good cytocompatibility at low concentrations, and in fact, the cell compatibility extends over a wider concentration range for Pam2CysOH. The TLR2/6 agonist activity was assessed using an assay that probes secreted alkaline phosphatase (SEAP) in NF-κB-SEAP reporter HEK293 cells expressing human TLR2 and TLR6, and Pam2CySOH shows significant activity, although not to the extent of Pam2CysSK4 or Pam3CysSK4. Thus, Pam2CysOH in particular is of interest as a vesicle-forming TLR2/6 agonist and stimulator of immune response.
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Apr 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Influenza virus neuraminidase is a crucial target for protective antibodies, yet the development of recombinant neuraminidase protein as a vaccine has been held back by instability and variable expression. We have taken a pragmatic approach to improving expression and stability of neuraminidase by grafting antigenic surface loops from low-expressing neuraminidase proteins onto the scaffold of high-expressing counterparts. The resulting hybrid proteins retained the antigenic properties of the loop donor while benefiting from the high-yield expression, stability, and tetrameric structure of the loop recipient. These hybrid proteins were recognised by a broad set of human monoclonal antibodies elicited by influenza infection or vaccination, with X-ray structures validating the accurate structural conformation of the grafted loops and the enzymatic cavity. Immunisation of mice with neuraminidase hybrids induced inhibitory antibodies to the loop donor and protected against lethal influenza challenge. This pragmatic technique offers a robust solution for improving the expression and stability of influenza neuraminidase proteins for vaccine development.
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Apr 2025
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Krios I-Titan Krios I at Diamond
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Lee
Sherry
,
Keith
Grehan
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Mohammad W.
Bahar
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Jessica J.
Swanson
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Helen
Fox
,
Sue
Matthews
,
Sarah
Carlyle
,
Ling
Qin
,
Claudine
Porta
,
Steven
Wilkinson
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Suzanne
Robb
,
Naomi
Clark
,
John
Liddell
,
Elizabeth E.
Fry
,
David I.
Stuart
,
Andrew J.
Macadam
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David J.
Rowlands
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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
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Mohammad W.
Bahar
,
Claudine
Porta
,
Helen
Fox
,
Keith
Grehan
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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
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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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B21-High Throughput SAXS
I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[27906, 37100]
Abstract: The storage and distribution of vital protein therapeutics presents several complex challenges. Many medicines and vaccines need stable, temperature-controlled environments and chemical additives (excipients) such as preservatives to keep them effective and safe for use.
This requires cold storage infrastructure and reliable energy sources which not only puts the responsibility on the user but causes accessibility and affordability challenges, especially in developing countries where resources are limited.
Fig. 1
Fig. 1
Now researchers from the UK Universities of Manchester, Glasgow and Warwick have designed the world’s first hydrogel technology for the storage and distribution of crucial medicines and other biopharmaceuticals without the need for refrigeration or chemical additives. The aim is to provide more robust and equitable storage and delivery systems, benefitting everyone worldwide.
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Oct 2024
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John D.
Clarke
,
Helen M. E.
Duyvesteyn
,
Eva
Perez-Martin
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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.
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Oct 2024
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Krios I-Titan Krios I at Diamond
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Victoria A.
Avanzato
,
Trenton
Bushmaker
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Kasopefoluwa Y.
Oguntuyo
,
Claude Kwe
Yinda
,
Helen M. E.
Duyvesteyn
,
Robert
Stass
,
Kimberly
Meade-White
,
Rebecca
Rosenke
,
Tina
Thomas
,
Neeltje
Van Doremalen
,
Greg
Saturday
,
Katie J.
Doores
,
Benhur
Lee
,
Thomas A.
Bowden
,
Vincent J.
Munster
Diamond Proposal Number(s):
[20223]
Abstract: Nipah virus (NiV) is a highly pathogenic paramyxovirus capable of causing severe respiratory and neurologic disease in humans. Currently, there are no licensed vaccines or therapeutics against NiV, underscoring the urgent need for the development of countermeasures. The NiV surface-displayed glycoproteins, NiV-G and NiV-F, mediate host cell attachment and fusion, respectively, and are heavily targeted by host antibodies. Here, we describe a vaccination-derived neutralizing monoclonal antibody, mAb92, that targets NiV-F. Structural characterization of the Fab region bound to NiV-F (NiV-F–Fab92) by cryo-electron microscopy analysis reveals an epitope in the DIII domain at the membrane distal apex of NiV-F, an established site of vulnerability on the NiV surface. Further, prophylactic treatment of hamsters with mAb92 offered complete protection from NiV disease, demonstrating beneficial activity of mAb92 in vivo. This work provides support for targeting NiV-F in the development of vaccines and therapeutics against NiV.
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Sep 2024
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