key: cord-0296234-gmejjkax authors: Wellens, J.; Edmans, M.; Obolski, U.; McGregor, C. G.; Simmonds, P.; Turner, M.; Jarvis, L.; Skelley, D.; Dunachie, S.; Eyre, D. W.; Colombel, J.-F.; Wong, S.-Y.; Klenerman, P.; Lindsey, J. O.; Satsangi, J.; Thompson, C. P. title: Combination therapy of infliximab and thiopurines, but not monotherapy with infliximab or vedolizumab, is associated with attenuated IgA and neutralisation responses to SARS-CoV-2 in inflammatory bowel disease. date: 2021-10-18 journal: nan DOI: 10.1101/2021.10.13.21264916 sha: 0b5382757959a14df396918bf11c1447ae0f50ae doc_id: 296234 cord_uid: gmejjkax There is substantial interest regarding the perceived risk that immunomodulator and biologic therapy could have on COVID-19 disease severity among patients with inflammatory bowel disease (IBD) and clinicians. In this study, we show that infliximab/thiopurine combination therapy is associated with significantly lower IgA, a range of lower IgG responses as well as impaired neutralising antibody responses, compared to responses observed in healthy individuals. We also demonstrate that whilst IgG responses were significantly reduced in individuals with IBD treated with infliximab or vedolizumab monotherapy compared to healthy controls, there was no significant reduction in IgA and neutralising antibody responses. As neutralising antibody responses correlate with protection, this observation may provide the mechanistic explanation for the observation reported by the SECURE-IBD study that individuals on infliximab/thiopurine combination therapy were at greater risk of severe COVID-19 outcomes than patients on monotherapy. antibody responses correlate with protection, this observation may provide the mechanistic explanation for the observation reported by the SECURE--IBD study that individuals on infliximab/thiopurine combination therapy were at greater risk of severe COVID--19 outcomes than patients on monotherapy. The effect of immunomodulator and biological therapy for inflammatory bowel disease (IBD) on the immune response to SARS--CoV--2 is of substantial interest to patients and clinicians worldwide. The CLARITY IBD study recently reported attenuated serological responses in IBD patients treated with infliximab in comparison to vedolizumab 1 , with the effect greatest in those on infliximab/thiopurine combination therapy. Independently, the global SECURE--IBD registry highlighted that infliximab/thiopurine combination therapy, but not infliximab or vedolizumab monotherapies, was associated with more severe clinical outcomes upon SARS--CoV--2 infection 2,3 . However, these studies have not addressed treatment effects on neutralising antibody responses, the key correlate of protection to SARS--CoV--2; nor have they analysed the range of serological signatures that may influence clinical outcomes 4,5 . To answer these questions, we performed an extended analysis of serological responses to SARS--CoV--2 infection in seropositive IBD patients treated with either infliximab or vedolizumab monotherapy, or infliximab/thiopurine combination therapy ( Figures 1&2 ). Blood samples were collected from consenting patients attending infusion centres in Oxford and London between May and December 2020. Sera were initially screened by Abbott assay for SARS--CoV--2 antibody responses 6 . Serological reactivity profiles in positive samples were compared with those from healthy adult controls seropositive in the same assay 7 (Supplementary information table 1) . Antibody reactivity to the receptor--binding domain (RBD) of the SARS--CoV--2 spike, full-length spike (S), and the nucleocapsid (N) was assayed by IgG/IgA standard enzyme-linked immunosorbent assays (ELISA) and IgG high--throughput MSD V--PLEX assay. An ACE2--SARS--CoV--2 RBD inhibition assay was used to detect neutralising antibodies 5,8 . All treatments were associated with significantly reduced IgG antibody responses compared to healthy controls for all SARS--CoV--2 antigens, using an MSD V--PLEX assay ( Figure 1 ). The greatest reduction in IgG response by ELISA was observed in individuals treated with infliximab/thiopurine combination therapy (Figure 2a ; p=0.00019). All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Furthermore, IgA responses were significantly reduced in individuals treated with infliximab/thiopurine combination therapy compared to healthy controls (Figure 2b; p=0.009), but not in IBD patients treated with infliximab or vedolizumab monotherapy. Next, we utilized an ELISA--based inhibition assay to determine the ability of serum to neutralize the binding of SARS--CoV--2 RBD--ACE2 interaction (Figure 2c ). Individuals treated with vedolizumab or infliximab monotherapy did not show a significant difference in neutralising antibody responses compared to healthy individuals ( Figure 2c ). However, individuals treated with infliximab/thiopurine combination therapy showed a significantly reduced response compared to either monotherapy groups, and to the healthy control group (Figure 2c , p=0.0054, 0.0022 and p= 0.0092). Our data are novel, firstly in demonstrating that infliximab/thiopurine combination therapy is associated with significantly lower IgA as well as a range of IgG responses, and most importantly, with impaired functional neutralising antibody responses, compared to responses in healthy individuals. Secondly, we show that whilst IgG responses were significantly reduced in individuals with IBD treated with infliximab or vedolizumab monotherapy compared to healthy controls, this was not the case for IgA and neutralising antibody responses. As neutralising antibody responses are demonstrated to correlate directly with protection (and inversely with severity 9,10 ), this observation may provide the mechanistic explanation for the observation reported by the SECURE--IBD study that individuals with combination therapy were at greater risk of severe COVID--19 outcomes than patients on monotherapy 9,10 . In demonstrating that these therapeutic interventions are selectively associated with a pattern of attenuated antibody responses to SARS--Cov2 infection compared to healthy controls, we believe these data extend current understanding in this important area, and have potentially important implications for patient care and vaccination strategies. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 18, 2021. Microbiol. 58, (2020). All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Pre--coated plates ('Coronavirus panel 2') were incubated at RT with Blocker A solution for at least 30 minutes whilst being shaken at 500--700 rpm. Serum or plasma was diluted in Diluent 100 at dilutions of 1:500 to 1:50,000 and samples were added to the plates in duplicate. Plates were incubated for 2 hours at RT, whilst being shaken at 500--700 rpm throughout. A 1x working concentration of the SULFO--TAG anti--human IgG Detection Antibody was prepared in Diluent 100. After incubation with the samples, the plates were washed x3 with 1x MSD Wash buffer. Prepared detection antibody solution was added to the plates, which were incubated at RT for 1 hour, whilst being shaken. Plates were then washed x3 with 1X MSD Wash buffer. To read the assay results, MSD GOLD Read Buffer B (provided ready to use) was added to the plate. No incubation is required, and the plates were read on a MESO QuickPlex SQ 120 (MSD, USA) immediately after adding the buffer. A 7--point calibration curve of the standards was prepared using Diluent 100. Diluent 100 was used as a negative control. An additional three positive controls provided with the kit were also run on every plate. All standards and controls were run in duplicate. Data from the assay was analysed using MSD Discovery Workbench software, which averaged all the duplicates, generated, and fitted the data to standard curves 3 . The qualitative immunoenzymatic determination of RBD--ACE2 inhibition antibodies is based on an ELISA based assay technique (The Native Antigen Company, Oxford, UK). Microplates were coated with RBD in Dulbecco's Phosphate Buffered Saline (DPBS) to bind corresponding ACE2 or blocking antibodies of the sample. After washing the wells with DPBS + 0.05% Tween 20 to remove all unbound sample material, serum or plasma samples were added at a 1:20 dilution and allowed to bind. After incubation a horseradish peroxidase (HRP) labelled ACE2 conjugate is added and incubated. This conjugate binds to the captured RBD which has not been bound by the antibody sample. In a second washing step, unbound conjugate is removed. Bound ACE2--HRP conjugate (that therefore represents the absence of neutralizing antibody), is visualized by adding TMB substrate. Sulphuric acid was added to stop the reaction. Absorbance at 450nm was then read using a GloMax microplate reader (Promega, USA). All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 18, 2021. ; https://doi.org/10.1101/2021.10.13.21264916 doi: medRxiv preprint This healthcare worker study 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, 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). None of our funding bodies had any role in study design Maintenance therapy with infliximab or vedolizumab in IBD is not associated with increased SARS--CoV--2 seroprevalence: UK experience in the 2020 pandemic Divergent trajectories of antiviral memory after SARS--Cov--2 infection Fatal COVID--19 Outcomes are Associated with an Antibody Response Targeting Epitopes Shared with Endemic Coronaviruses JS has received lecture fees from Takeda and from the Falk Foundation.