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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 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 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.Publication Metadata only Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum(2021-08-05) Chang Liu; Helen M. Ginn; Wanwisa Dejnirattisai; Piyada Supasa; Beibei Wang; Aekkachai Tuekprakhon; Rungtiwa Nutalai; Daming Zhou; Alexander J. Mentzer; Yuguang Zhao; Helen M.E. Duyvesteyn; César López-Camacho; Jose Slon-Campos; Thomas S. Walter; Donal Skelly; Sile Ann Johnson; Thomas G. Ritter; Chris Mason; 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; Nigel Temperton; 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; Tariq Malik; Miles W. Carroll; Paul Klenerman; Eleanor Barnes; Susanna J. Dunachie; Vicky Baillie; Natali Serafin; Zanele Ditse; Kelly Da Silva; Neil G. Paterson; Mark A. Williams; David R. Hall; Shabir Madhi; Marta C. Nunes; Philip Goulder; 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; Texas A&M College of Veterinary Medicine & Biomedical Sciences; Public Health England; Diamond Light Source; University of the Witwatersrand Faculty of Health Sciences; University of Oxford; Fundacao Oswaldo Cruz; Fiocruz Amazônia; School of Pathology; University of Kent; Nuffield Department of Medicine; Università degli Studi di Siena; University of Oxford Medical Sciences Division; Oxford House; Fundação de Vigilância em Saúde do AmazonasSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone progressive change, with variants conferring advantage rapidly becoming dominant lineages, e.g., B.1.617. With apparent increased transmissibility, variant B.1.617.2 has contributed to the current wave of infection ravaging the Indian subcontinent and has been designated a variant of concern in the United Kingdom. Here we study the ability of monoclonal antibodies and convalescent and vaccine sera to neutralize B.1.617.1 and B.1.617.2, complement this with structural analyses of Fab/receptor binding domain (RBD) complexes, and map the antigenic space of current variants. Neutralization of both viruses is reduced compared with ancestral Wuhan-related strains, but there is no evidence of widespread antibody escape as seen with B.1.351. However, B.1.351 and P.1 sera showed markedly more reduction in neutralization of B.1.617.2, suggesting that individuals infected previously by these variants may be more susceptible to reinfection by B.1.617.2. This observation provides important new insights for immunization policy with future variant vaccines in non-immune populations.Publication Metadata only Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19(2020-01-01) Yanchun Peng; Alexander J. Mentzer; Guihai Liu; Xuan Yao; Zixi Yin; Danning Dong; Wanwisa Dejnirattisai; Timothy Rostron; Piyada Supasa; Chang Liu; César López-Camacho; Jose Slon-Campos; Yuguang Zhao; David I. Stuart; Guido C. Paesen; Jonathan M. Grimes; Alfred A. Antson; Oliver W. Bayfield; Dorothy E.D.P. Hawkins; De Sheng Ker; Beibei Wang; Lance Turtle; Krishanthi Subramaniam; Paul Thomson; Ping Zhang; Christina Dold; Jeremy Ratcliff; Peter Simmonds; Thushan de Silva; Paul Sopp; Dannielle Wellington; Ushani Rajapaksa; Yi Ling Chen; Mariolina Salio; Giorgio Napolitani; Wayne Paes; Persephone Borrow; Benedikt M. Kessler; Jeremy W. Fry; Nikolai F. Schwabe; Malcolm G. Semple; J. Kenneth Baillie; Shona C. Moore; Peter J.M. Openshaw; M. Azim Ansari; Susanna Dunachie; Eleanor Barnes; John Frater; Georgina Kerr; Philip Goulder; Teresa Lockett; Robert Levin; Yonghong Zhang; Ronghua Jing; Ling Pei Ho; Eleanor Barnes; Danning Dong; Tao Dong; Susanna Dunachie; John Frater; Philip Goulder; Georgina Kerr; Paul Klenerman; Guihai Liu; Andrew McMichael; Graham Ogg; Yanchun Peng; Mariolina Salio; Xuan Yao; Zixi Yin; J. Kenneth Baillie; Paul Klenerman; Alexander J. Mentzer; Shona C. Moore; Peter J.M. Openshaw; Malcolm G. Semple; David I. Stuart; Richard J. Cornall; Christopher P. Conlon; Paul Klenerman; Gavin R. Screaton; Juthathip Mongkolsapaya; Andrew McMichael; Julian C. Knight; Graham Ogg; Tao Dong; NIHR Oxford Biomedical Research Centre; Oxford University Hospitals NHS Foundation Trust; Liverpool University Hospitals NHS Foundation Trust; Beijing YouAn Hospital, Capital Medical University; Diamond Light Source; The Wellcome Centre for Human Genetics; Worthing Hospital; The University of Edinburgh; University of Oxford; Alder Hey Children's Hospital; Xinjiang Medical University; University of Liverpool; University of York; Faculty of Medicine, Siriraj Hospital, Mahidol University; National Heart and Lung Institute; Nuffield Department of Medicine; The Sheffield Medical School; University of Oxford Medical Sciences Division; ProImmune© 2020, The Author(s), under exclusive licence to Springer Nature America, Inc. The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide–MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.