Structural epitope profiling identifies antibodies associated with critical COVID-19 and long COVID

Antibodies can have beneficial, neutral, or harmful effects so resolving an antibody repertoire to its target epitopes may explain heterogeneity in susceptibility to infectious disease. However, the three-dimensional nature of antibody-epitope interactions limits discovery of important targets. We describe and experimentally validated a novel computational method and synthetic biology pipeline for identifying epitopes that are structurally stable and functionally important and apply it to the SARS-CoV-2 proteome. We show patterns of epitope-binding antibodies associated with immunopathology, including a non-isotype switching IgM response to a Membrane protein epitope which is amongst the strongest immunological features associated with severe COVID-19 to date (adjusted OR 72.14, 95% CI: 9.71 – 1300.15). Consistent with a hypothesis that the mechanism driving the non-switching response was T independent B cell activation, we find that B cells secrete IgM and proliferate on exposure to virus-like particles lacking Spike. We also identified persistence (> 1 year) of this response in individuals with longCOVID particularly affected by fatigue and depression. These findings point to a previously unrecognized coronavirus host-pathogen interaction. We demonstrate that the Membrane epitope is a promising vaccine and monoclonal antibody target, which may complement spike-directed vaccination broadening immunological protection. One-Sentence Summary Using a protein-structure-based B cell epitope discovery method with a wide range of possible applications, we have identified a novel host-pathogen signature associated with SARS-CoV-2 immunopathology and suggest the viral Membrane protein contains a pathological T independent antigen. ### Competing Interest Statement PKAK, DJK, JAM, and NG are inventors on a preliminary application for a patent filed 9th December 2021 by University of Edinburgh titled "Thermodynamic prediction, synthesis and prioritisation of immunogenic peptides" and have no other competing interests. AC is a paid editor of JNNP, unpaid president of functional neurological disorders society and gives independent testimony in court on a range of neuropsychiatric subjects on a roughly 50% claimant 50% defender split. LMcW is unpaid as a secretary of British Neuropsychiatry Association and gives independent testimony on court on a range of neuropsychiatric subjects. TL is named as an inventor on a patent application for a vaccine against SARS CoV-2 for an unrelated project. TL was a consultant to Vaccitech for an unrelated project. AJP is chair of the UK Department of Health and Social Care's Joint Committee on Vaccination and Immunisation but does not participate in the JCVI COVID19 committee. He was previously a member of WHO's SAGE. The University of Oxford has a partnership with AstraZeneca for the development of COVID19 vaccines. Other authors declare that they have no competing interests. ### Funding Statement PKAK. is supported by a ECAT-Wellcome fellowship (223058/Z/21/Z). NG is supported by MRC (MC\_UU\_00007/13). JAM is a Lister Institute Research Fellow. Lifearc, UKRI, Medical Research Scotland provided funding for development of the method. The long COVID study was funded by the chief Scientist Office Scotland. The CIRCO (Manchester) study was funded by the Wellcome Trust (202865/Z/16/Z). The Oxford cohort was funded by the UK Department of Health and Social Care as part of the PITCH (Protective Immunity from T cells to Covid-19 in Health workers) Consortium, UKRI as part of "Investigation of proven vaccine breakthrough by SARS-CoV-2 variants in established UK healthcare worker cohorts: SIREN consortium & PITCH Plus Pathway" MR/W02067X/1, with contributions from UKRI/NIHR through the UK Coronavirus Immunology Consortium (UK-CIC), the Huo Family Foundation and The National Institute for Health Research (UKRIDHSC COVID-19 Rapid Response Rolling Call, Grant Reference Number COV19-RECPLAS) and the UK Vaccine Taskforce via NIHR grant to support the running of the Oxford ChAdOx1 nCoV-19 vaccine trial paid to the University of Oxford. JK was funded by UKRI (MR/W020629/1) E.B. and P.K. are NIHR Senior Investigators and P.K. is funded by WT109965MA. S.J.D. is funded by an NIHR Global Research Professorship (NIHR300791). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: North of Scotland Research Ethics Committee of the National Health Service (Scotland) gave ethical approval for the long COVID study (ref 21/NS/0035). The Research and Sample Governance Committee of the Scottish National Blood Transfusion Service gave ethical approval for the work involving blood donors (ref: SG2021/25). The Oxfordshire Research Ethics Committee C of National Health Services Health Research Authority gave ethical approval for the work involving subjects with severe COVID-19 (Oxford REC C, reference:19/SC/0296 and Oxford REC C, reference 13/SC/0149). The Northwest-Preston Research Ethics Committee of National Health Services Health Research Authority gave ethical approval for this work (Northwest-Preston REC, reference 20/NW/0235). The Sheffield Research Ethics Committee of National Health Services Health Research Authority gave ethical approval for the work recruiting recovered HCWs under the Gastrointestinal illness in Oxford: COVID sub study (ref: 16/YH/0247). The London-Brent Research Ethics Committee of the National Health Service (Scotland) gave ethical approval for this work. (NHS Lothian BioResource (SR1407) and ref: (ref: 20/HRA/3764 IRAS:28653)). The North West Haydock Research Ethics Committee of National Health Services Health Research Authority gave ethical approval for the work (ref: 15/NW/0409 for ManARTS). The Wales Research Ethics Committee 4 of Northern Care Alliance NHS Foundation Trust gave ethical approval for this work (ref: 18/WA/0368). The Edinburgh Medical School Research Ethics Committee of the University of Edinburgh gave ethical approval for this work (ref: 21/EMREC/010). All participants gave written and informed consent for serial blood sample collection. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data necessary are available in the text or the supplementary materials. Code to reproduce the analyses in the paper can be accessed from the first author's GitHub project repository. [https://github.com/PKKearns/SARS2\_Antibody\_Profiling_Paper][1] [1]: https://github.com/PKKearns/SARS2_Antibody_Profiling_Paper