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Publication Metadata only Flavivirus maturation leads to the formation of an occupied lipid pocket in the surface glycoproteins(2021-12-01) Max Renner; Wanwisa Dejnirattisai; Loïc Carrique; Itziar Serna Martin; Dimple Karia; Serban L. Ilca; Shu F. Ho; Abhay Kotecha; Jeremy R. Keown; Juthathip Mongkolsapaya; Gavin R. Screaton; Jonathan M. Grimes; Siriraj Hospital; Diamond Light Source; The Wellcome Centre for Human Genetics; Bijvoet Centre for Biomolecular Research; Nuffield Department of MedicineFlaviviruses such as Dengue (DENV) or Zika virus (ZIKV) assemble into an immature form within the endoplasmatic reticulum (ER), and are then processed by furin protease in the trans-Golgi. To better grasp maturation, we carry out cryo-EM reconstructions of immature Spondweni virus (SPOV), a human flavivirus of the same serogroup as ZIKV. By employing asymmetric localised reconstruction we push the resolution to 3.8 Å, enabling us to refine an atomic model which includes the crucial furin protease recognition site and a conserved Histidine pH-sensor. For direct comparison, we also solve structures of the mature forms of SPONV and DENV to 2.6 Å and 3.1 Å, respectively. We identify an ordered lipid that is present in only the mature forms of ZIKV, SPOV, and DENV and can bind as a consequence of rearranging amphipathic stem-helices of E during maturation. We propose a structural role for the pocket and suggest it stabilizes mature E.Publication Metadata only Characterization of a potent and highly unusual minimally enhancing antibody directed against dengue virus(2018-11-01) Max Renner; Aleksandra Flanagan; Wanwisa Dejnirattisai; Chunya Puttikhunt; Watchara Kasinrerk; Piyada Supasa; Wiyada Wongwiwat; Kriangkrai Chawansuntati; Thaneeya Duangchinda; Alison Cowper; Claire M. Midgley; Prida Malasit; Juha T. Huiskonen; Juthathip Mongkolsapaya; Gavin R. Screaton; Jonathan M. Grimes; Diamond Light Source; Wellcome Trust Centre for Human Genetics; Faculty of Medicine, Siriraj Hospital, Mahidol University; Nuffield Department of Clinical Medicine; Chiang Mai University© 2018, The Author(s), under exclusive licence to Springer Nature America, Inc. Dengue virus is a major pathogen, and severe infections can lead to life-threatening dengue hemorrhagic fever. Dengue virus exists as four serotypes, and dengue hemorrhagic fever is often associated with secondary heterologous infections. Antibody-dependent enhancement (ADE) may drive higher viral loads in these secondary infections and is purported to result from antibodies that recognize dengue virus but fail to fully neutralize it. Here we characterize two antibodies, 2C8 and 3H5, that bind to the envelope protein. Antibody 3H5 is highly unusual as it not only is potently neutralizing but also promotes little if any ADE, whereas antibody 2C8 has strong capacity to promote ADE. We show that 3H5 shows resilient binding in endosomal pH conditions and neutralizes at low occupancy. Immunocomplexes of 3H5 and dengue virus do not efficiently interact with Fcγ receptors, which we propose is due to the binding mode of 3H5 and constitutes the primary mechanism of how ADE is avoided.Publication Metadata only Structural analysis of a dengue cross-reactive antibody complexed with envelope domain III reveals the molecular basis of cross-reactivity(2012-05-15) Claire M. Midgley; Aleksandra Flanagan; Hai Bac Tran; Wanwisa Dejnirattisai; Kriangkrai Chawansuntati; Amonrat Jumnainsong; Wiyada Wongwiwat; Thaneeya Duangchinda; Juthathip Mongkolsapaya; Jonathan M. Grimes; Gavin R. Screaton; Hammersmith Hospital; Wellcome Trust Centre for Human Genetics; Thailand National Center for Genetic Engineering and Biotechnology; Mahidol University; Diamond Light SourceDengue virus infections are still increasing at an alarming rate in tropical and subtropical countries, underlying the need for a dengue vaccine. Although it is relatively easy to generate Ab responses to dengue virus, low avidity or low concentrations of Ab may enhance infection of FcR-bearing cells with clinical impact, posing a challenge to vaccine production. In this article, we report the characterization of a mAb, 2H12, which is cross-reactive to all four serotypes in the dengue virus group. Crystal structures of 2H12- Fab in complex with domain III of the envelope protein from three dengue serotypes have been determined. 2H12 binds to the highly conserved AB loop of domain III of the envelope protein that is poorly accessible in the mature virion. 2H12 neutralization varied between dengue serotypes and strains; in particular, dengue serotype 2 was not neutralized. Because the 2H12-binding epitope was conserved, this variation in neutralization highlights differences between dengue serotypes and suggests that significant conformational changes in the virus must take place for Ab binding. Surprisingly, 2H12 facilitated little or no enhancement of infection. These data provide a structural basis for understanding Ab neutralization and enhancement of infection, which is crucial for the development of future dengue vaccines. Copyright © 2012 by The American Association of Immunologists, Inc.Publication Metadata only Native-like SARS-CoV-2 Spike Glycoprotein Expressed by ChAdOx1 nCoV-19/AZD1222 Vaccine(2021-04-28) Yasunori Watanabe; Luiza Mendonça; Elizabeth R. Allen; Andrew Howe; Mercede Lee; Joel D. Allen; Himanshi Chawla; David Pulido; Francesca Donnellan; Hannah Davies; Marta Ulaszewska; Sandra Belij-Rammerstorfer; Susan Morris; Anna Sophia Krebs; Wanwisa Dejnirattisai; Juthathip Mongkolsapaya; Piyada Supasa; Gavin R. Screaton; Catherine M. Green; Teresa Lambe; Peijun Zhang; Sarah C. Gilbert; Max Crispin; Siriraj Hospital; NIHR Oxford Biomedical Research Centre; Diamond Light Source; The Wellcome Centre for Human Genetics; University of Oxford; University of Southampton; Nuffield Department of Medicine; University of Oxford Medical Sciences DivisionVaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation, and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirm the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.Publication Metadata only Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike(2020-01-01) Jiandong Huo; Yuguang Zhao; Jingshan Ren; Daming Zhou; Helen M.E. Duyvesteyn; Helen M. Ginn; Loic Carrique; Tomas Malinauskas; Reinis R. Ruza; Pranav N.M. Shah; Tiong Kit Tan; Pramila Rijal; Naomi Coombes; Kevin R. Bewley; Julia A. Tree; Julika Radecke; Neil G. Paterson; Piyasa Supasa; Juthathip Mongkolsapaya; Gavin R. Screaton; Miles Carroll; Alain Townsend; Elizabeth E. Fry; Raymond J. Owens; David I. Stuart; Public Health England; Diamond Light Source; The Wellcome Centre for Human Genetics; University of Oxford; Faculty of Medicine, Siriraj Hospital, Mahidol University; Nuffield Department of Medicine; MRC Weatherall Institute of Molecular Medicine; Rosalind Franklin Institute; INSTRUCT-ERIC; Research Complex at Harwell© 2020 There are as yet no licensed therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2, initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralizing SARS-CoV-2, and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilizing CR3022 epitope is inaccessible in the prefusion spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryogenic electron microscopy (cryo-EM) analysis confirms that incubation of spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope could be useful therapeutically, possibly in synergy with an antibody that blocks receptor attachment.Publication Metadata only Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera(2021-04-15) Piyada Supasa; Daming Zhou; Wanwisa Dejnirattisai; Chang Liu; Alexander J. Mentzer; Helen M. Ginn; Yuguang Zhao; Helen M.E. Duyvesteyn; Rungtiwa Nutalai; Aekkachai Tuekprakhon; Beibei Wang; Guido C. Paesen; Jose Slon-Campos; César López-Camacho; Bassam Hallis; Naomi Coombes; Kevin R. Bewley; Sue Charlton; Thomas S. Walter; Eleanor Barnes; Susanna J. Dunachie; Donal Skelly; Sheila F. Lumley; Natalie Baker; Imam Shaik; Holly E. Humphries; Kerry Godwin; Nick Gent; Alex Sienkiewicz; Christina Dold; Robert Levin; Tao Dong; Andrew J. Pollard; Julian C. Knight; Paul Klenerman; Derrick Crook; Teresa Lambe; Elizabeth Clutterbuck; Sagida Bibi; Amy Flaxman; Mustapha Bittaye; Sandra Belij-Rammerstorfer; Sarah Gilbert; David R. Hall; Mark A. Williams; Neil G. Paterson; William James; Miles W. Carroll; Elizabeth E. Fry; Juthathip Mongkolsapaya; Jingshan Ren; David I. Stuart; Gavin R. Screaton; Mahidol Oxford Tropical Medicine Research Unit; NIHR Oxford Biomedical Research Centre; Oxford University Hospitals NHS Foundation Trust; Public Health England; Diamond Light Source; Worthing Hospital; University of Oxford; Sir William Dunn School of Pathology; Faculty of Medicine Siriraj Hospital, Mahidol University; Nuffield Department of Medicine; University of Oxford Medical Sciences Division; Instruct-ERIC, compromising neutralization by some members of a major class of public antibodies through light-chain contacts with residue 501. However, widespread escape from monoclonal antibodies or antibody responses generated by natural infection or vaccinationPublication Metadata only Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera(2021-04-29) Daming Zhou; Wanwisa Dejnirattisai; Piyada Supasa; Chang Liu; Alexander J. Mentzer; Helen M. Ginn; Yuguang Zhao; Helen M.E. Duyvesteyn; Aekkachai Tuekprakhon; Rungtiwa Nutalai; Beibei Wang; Guido C. Paesen; Cesar Lopez-Camacho; Jose Slon-Campos; Bassam Hallis; Naomi Coombes; Kevin Bewley; Sue Charlton; Thomas S. Walter; Donal Skelly; Sheila F. Lumley; Christina Dold; Robert Levin; Tao Dong; Andrew J. Pollard; Julian C. Knight; Derrick Crook; Teresa Lambe; Elizabeth Clutterbuck; Sagida Bibi; Amy Flaxman; Mustapha Bittaye; Sandra Belij-Rammerstorfer; Sarah Gilbert; William James; Miles W. Carroll; Paul Klenerman; Eleanor Barnes; Susanna J. Dunachie; Elizabeth E. Fry; Juthathip Mongkolsapaya; Jingshan Ren; David I. Stuart; Gavin R. Screaton; Siriraj Hospital; Mahidol Oxford Tropical Medicine Research Unit; NIHR Oxford Biomedical Research Centre; Oxford University Hospitals NHS Foundation Trust; Public Health England; Diamond Light Source; Worthing Hospital; University of Oxford; Sir William Dunn School of Pathology; Nuffield Department of Medicine; University of Oxford Medical Sciences Division; Instruct-ERICThe race to produce vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began when the first sequence was published, and this forms the basis for vaccines currently deployed globally. Independent lineages of SARS-CoV-2 have recently been reported: UK, B.1.1.7; South Africa, B.1.351; and Brazil, P.1. These variants have multiple changes in the immunodominant spike protein that facilitates viral cell entry via the angiotensin-converting enzyme-2 (ACE2) receptor. Mutations in the receptor recognition site on the spike are of great concern for their potential for immune escape. Here, we describe a structure-function analysis of B.1.351 using a large cohort of convalescent and vaccinee serum samples. The receptor-binding domain mutations provide tighter ACE2 binding and widespread escape from monoclonal antibody neutralization largely driven by E484K, although K417N and N501Y act together against some important antibody classes. In a number of cases, it would appear that convalescent and some vaccine serum offers limited protection against this variant.Publication Metadata only Antibody evasion by the P.1 strain of SARS-CoV-2(2021-05-27) Wanwisa Dejnirattisai; Daming Zhou; Piyada Supasa; Chang Liu; Alexander J. Mentzer; Helen M. Ginn; Yuguang Zhao; Helen M.E. Duyvesteyn; Aekkachai Tuekprakhon; Rungtiwa Nutalai; Beibei Wang; César López-Camacho; Jose Slon-Campos; Thomas S. Walter; Donal Skelly; Sue Ann Costa Clemens; Felipe Gomes Naveca; Valdinete Nascimento; Fernanda Nascimento; Cristiano Fernandes da Costa; Paola Cristina Resende; Alex Pauvolid-Correa; Marilda M. Siqueira; Christina Dold; Robert Levin; Tao Dong; Andrew J. Pollard; Julian C. Knight; Derrick Crook; Teresa Lambe; Elizabeth Clutterbuck; Sagida Bibi; Amy Flaxman; Mustapha Bittaye; Sandra Belij-Rammerstorfer; Sarah C. Gilbert; Miles W. Carroll; Paul Klenerman; Eleanor Barnes; Susanna J. Dunachie; Neil G. Paterson; Mark A. Williams; David R. Hall; Ruben J.G. Hulswit; Thomas A. Bowden; Elizabeth E. Fry; Juthathip Mongkolsapaya; Jingshan Ren; David I. Stuart; Gavin R. Screaton; Siriraj Hospital; NIHR Oxford Biomedical Research Centre; Oxford University Hospitals NHS Foundation Trust; Texas A&M College of Veterinary Medicine & Biomedical Sciences; Public Health England; Diamond Light Source; Worthing Hospital; University of Oxford; Fundacao Oswaldo Cruz; Fiocruz Amazônia; Nuffield Department of Medicine; University of Oxford Medical Sciences Division; Instruct-ERIC; Fundação de Vigilância em Saúde do Amazonas(RBD) impact neutralization. Monoclonal antibody (mAb) 222 neutralizes all three variants despite interacting with two of the ACE2-binding site mutations. We explain this through structural analysis and use the 222 light chain to largely restorePublication Metadata only Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient(2020-01-01) Daming Zhou; Helen M.E. Duyvesteyn; Cheng Pin Chen; Chung Guei Huang; Ting Hua Chen; Shin Ru Shih; Yi Chun Lin; Chien Yu Cheng; Shu Hsing Cheng; Yhu Chering Huang; Tzou Yien Lin; Che Ma; Jiandong Huo; Loic Carrique; Tomas Malinauskas; Reinis R. Ruza; Pranav N.M. Shah; Tiong Kit Tan; Pramila Rijal; Robert F. Donat; Kerry Godwin; Karen R. Buttigieg; Julia A. Tree; Julika Radecke; Neil G. Paterson; Piyada Supasa; Juthathip Mongkolsapaya; Gavin R. Screaton; Miles W. Carroll; Javier Gilbert-Jaramillo; Michael L. Knight; William James; Raymond J. Owens; James H. Naismith; Alain R. Townsend; Elizabeth E. Fry; Yuguang Zhao; Jingshan Ren; David I. Stuart; Kuan Ying A. Huang; Public Health England; Diamond Light Source; Academia Sinica, Genomics Research Center; National Yang-Ming University Taiwan; Chang Gung Memorial Hospital; University of Oxford; Taipei Medical University; Chang Gung University; Sir William Dunn School of Pathology; Faculty of Medicine, Siriraj Hospital, Mahidol University; Nuffield Department of Medicine; MRC Weatherall Institute of Molecular Medicine; The Rosalind Franklin Institute; Research Complex© 2020, The Author(s), under exclusive licence to Springer Nature America, Inc. The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (KD of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD–EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.Publication Metadata only The antigenic anatomy of SARS-CoV-2 receptor binding domain(2021-04-15) Wanwisa Dejnirattisai; Daming Zhou; Helen M. Ginn; Helen M.E. Duyvesteyn; Piyada Supasa; James Brett Case; Yuguang Zhao; Thomas S. Walter; Alexander J. Mentzer; Chang Liu; Beibei Wang; Guido C. Paesen; Jose Slon-Campos; César López-Camacho; Natasha M. Kafai; Adam L. Bailey; Rita E. Chen; Baoling Ying; Craig Thompson; Jai Bolton; Alex Fyfe; Sunetra Gupta; Tiong Kit Tan; Javier Gilbert-Jaramillo; William James; Michael Knight; Miles W. Carroll; Donal Skelly; Christina Dold; Yanchun Peng; Robert Levin; Tao Dong; Andrew J. Pollard; Julian C. Knight; Paul Klenerman; Nigel Temperton; David R. Hall; Mark A. Williams; Neil G. Paterson; Felicity K.R. Bertram; C. Alistair Siebert; Daniel K. Clare; Andrew Howe; Julika Radecke; Yun Song; Alain R. Townsend; Kuan Ying A. Huang; Elizabeth E. Fry; Juthathip Mongkolsapaya; Michael S. Diamond; Jingshan Ren; David I. Stuart; Gavin R. Screaton; NIHR Oxford Biomedical Research Centre; Oxford University Hospitals NHS Foundation Trust; Public Health England; Diamond Light Source; Chang Gung University College of Medicine; Chang Gung Memorial Hospital; Worthing Hospital; University of Oxford; Washington University School of Medicine in St. Louis; Sir William Dunn School of Pathology; Faculty of Medicine Siriraj Hospital, Mahidol University; University of Kent; Nuffield Department of Medicine; University of Oxford Medical Sciences Division; Instruct-ERICAntibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 μg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.