key: cord-0969501-jsrhzdcv authors: Davis, S. K.; Selva, K.; Lopez, E.; Haycroft, E.; Lee, W. S.; Wheatley, A. K.; Juno, J. A.; Adair, A.; Pymm, P.; Redmond, S.; Gherardin, N.; Godfrey, D.; Tham, W.-H.; Kent, S.; Chung, A. title: Heterologous SARS-CoV-2 IgA neutralising antibody responses in convalescent plasma date: 2022-02-08 journal: nan DOI: 10.1101/2022.02.06.22270359 sha: fdc5ae74bb9ee5b0a0302a59267cf02fbcfa81bf doc_id: 969501 cord_uid: jsrhzdcv Following infection with SARS-CoV-2, virus-specific antibodies are generated which can both neutralise virions and clear infection via Fc effector functions. The importance of IgG antibodies for protection and control of SARS-CoV-2 has been extensively reported. In comparison, other antibody isotypes including IgA have been poorly characterized. Here we characterized plasma IgA from 41 early convalescent COVID-19 subjects for neutralisation and Fc effector functions. We find that convalescent plasma IgA from >60% of the cohort have the capacity to inhibit the interaction between wild-type RBD and ACE2. Furthermore, a third of the cohort induced stronger IgA-mediated inhibition of RBD binding to ACE2 than IgG, when tested at equivalent concentrations. Plasma IgA and IgG from the cohort, broadly recognize similar RBD epitopes and showed similar ability to inhibit ACE2 from binding 22 of 23 different prevalent RBD proteins with single amino acid mutations. Plasma IgA was largely incapable of mediating antibody-dependent phagocytosis in comparison to plasma IgG. Overall, convalescent plasma IgA contributes to neutralisation towards wild-type RBD and various RBD single mutants in most subjects, although this response is heterogeneous and less potent than IgG. characterized plasma IgA from 41 early convalescent COVID-19 subjects for neutralisation and 23 Fc effector functions. We find that convalescent plasma IgA from >60% of the cohort have the 24 capacity to inhibit the interaction between wild-type RBD and ACE2. Furthermore, a third of 25 the cohort induced stronger IgA-mediated inhibition of RBD binding to ACE2 than IgG, when 26 tested at equivalent concentrations. Plasma IgA and IgG from the cohort, broadly recognize 27 similar RBD epitopes and showed similar ability to inhibit ACE2 from binding 22 of 23 different 28 prevalent RBD proteins with single amino acid mutations. Plasma IgA was largely incapable of 29 mediating antibody-dependent phagocytosis in comparison to plasma IgG. Overall, 30 convalescent plasma IgA contributes to neutralisation towards wild-type RBD and various RBD 31 single mutants in most subjects, although this response is heterogeneous and less potent than 32 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of 39 Coronavirus disease 2019 (COVID-19) has infected millions of people and caused over 5.6 40 million deaths globally since its discovery. SARS-CoV-2 trimeric spike protein consists of two 41 domains; spike 1 (S1) and spike 2 (S2) (Wrapp et al., 2020) . The receptor binding domain (RBD) 42 within the S1 engages with angiotensin-converting enzyme 2 (ACE2) on human cells 43 contributing to infection (Wrapp et al., 2020) . Antibodies generated towards RBD To examine the ability of subject plasma to neutralise SARS-CoV-2, we used a multiplex 100 RBDWT-ACE2 binding inhibition assay, that has been demonstrated to highly correlate with 101 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint . 1d ). Significant S1-ACE2 binding inhibition was also observed 105 for convalescent subject plasma compared to uninfected subjects (sup. fig. 1d & e) . Thus, 106 most SARS-CoV-2 convalescent plasma induced antibodies that recognise RBDWT and have 107 the capacity to inhibit the binding of RBD to ACE2, as previously reported (Lopez et al., 2021 . 1g ). This was further confirmed by individual correlations with anti-RBDWT 126 antibody isotypes (sup. fig. 2 ). Similar trends were observed for S1 (sup. fig. 1a To define the contribution of IgG and IgA to the neutralising 136 capacity of convalescent plasma, we depleted IgA from plasma (IgA-plasma) and also 137 depleted plasma of both IgA and IgG (IgA-and IgG-depleted plasma) to assess the capacity 138 of antibody depleted plasma fractions to inhibit RBDWT binding to ACE2 ( fig. 2, sup. fig. 3 ). 139 Successful IgG and IgA depletion was confirmed by a significant reduction in IgA and IgG 140 binding to RBDWT (p<0.0001) and S1 (p<0.0001) for IgA-and IgA-/IgG-plasma (sup. fig. 3f Although the above depletion studies suggest IgA contributes to convalescent plasma 159 neutralisation in the majority of the cohort, a more definitive role of IgA or IgG in 160 neutralisation could be revealed by specifically purifying these fractions and measuring 161 antibody binding using a multiplex bead array. First, we investigated the capacity for purified 162 IgG and IgA to mediate ACE2 binding inhibition to RBDWT at 100μg/ml total antibody. Purified 163 convalescent IgG (median=27.01% IQR 13-37%-41.87%) and IgA (median=12.17% IQR 0-164 31.39) mediated increased RBDWT-ACE2 binding inhibition compared to purified IgA 165 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint to the low concentration of purified IgA (100μg/ml) antibody available for use in these assays. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 8, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 8, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 8, 2022. significantly alter the cell association mediated by convalescent plasma (median 293 association=15.04% association IQR= 6.63%-27.66%, p>0.99) ( fig.4d ). However, depletion of 294 IgG (IgA-/IgG-plasma) (median association=1.33% association IQR=0.61%-3.10%) resulted in 295 a median loss of 90.53% cell association relative to convalescent plasma (median 296 association=15.04% association IQR= 6.63%-27.66%, p<0.0001) ( fig. 4d ). This data further 297 supports the role of IgG convalescent plasma for an effective Fc functional response to SARS- is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint single amino acid RBD mutants compared to IgG. We observed minor differences in purified 360 antibody isotype binding to various RBD single mutants, despite the small sample sizes and 361 the polyclonal nature of these convalescent responses. A trend toward preferential IgG 362 binding to many mutants was observed, with significantly increased IgG binding to 5 363 mutations. Interestingly, we observed a trend towards increased IgA binding to a single RBD 364 mutation; G446V, which is predicted to reduce RBD stability and increase ACE2 affinity 365 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Overall, we find that convalescent plasma IgA recognises RBDWT and an array of RBD mutants 388 and has the capacity to block ACE2 engagement with RBD in a comparable manner to IgG, 389 when sufficient IgA titers are induced. Furthermore, the convalescent IgA neutralising 390 response is highly heterogenous between individuals, with a third of the cohort inducing 391 stronger IgA-mediated inhibition of RBD engagement with ACE2 than IgG, when tested at 392 equivalent concentrations. Dissecting the IgA response in the context of vaccination and to 393 variants of concern is essential to further understand the importance of IgA in a protective 394 polyclonal antibody response. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 8, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 8, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Anti-RBDWT IgM (sup. fig. 3c ) and IgG (sup. fig. 3d ) binding was also assessed for plasma, IgA-487 and IgA-/IgG-depleted plasma via multiplex. During the depletion process, a loss of 48.88% 488 of IgG (sup. fig. 3c, median MFI=21150, p=0 .0160) and 36.95% of IgM (sup. fig. 3d, median 489 MFI=26544, p=0.0002) between medians was observed following the depletion of IgA (IgA-490 plasma) and IgG (IgA-/IgG-plasma) respectively compared to plasma (median IgG MFI=41377, 491 median IgM MFI=42101) (sup. fig. 3c-d) . To ensure fair comparisons between plasma and 492 depleted plasma fractions, plasma and depleted plasma samples were titrated, and anti-493 RBDWT IgG and IgM binding MFI was determined via multiplex (process outlined in sup. fig. 494 3e). IgA-and IgA-/IgG-depleted plasma dilutions were chosen by matching IgG or IgM MFI 495 respectively to plasma IgG or IgM binding MFI at a dilution of 1:100. IgA-plasma and IgA-/IgG-496 depleted plasma with final dilutions outlined in sup. table 2. These dilutions were used for all 497 multiplex assays. Depleted samples with >30% loss in IgG or IgM following matching of 498 dilutions were excluded from this study (sup. fig. 3b ). 499 500 Quality control testing of antibody depleted plasma 501 Successful depletion IgG and IgA was confirmed for matched dilutions via IgG SARS-CoV-2 502 RBDWT multiplex (sup. fig. 3f -k, sup. table 2). This method detected the remaining antigen 503 specific IgG or IgA with high sensitivity which allowed for application of a stringent threshold 504 for exclusion of samples with un-successful IgG or IgA depletion. Anti-RBDWT IgG or IgA 505 binding MFI of depleted plasma was compared to full plasma for each subject and the 506 percentage reduction in IgG and IgA binding was calculated Sufficient depletion was defined 507 as >70% depletion of IgG or IgA (i.e. >70% reduction in anti-RBDWT IgG MFI compared to 508 plasma IgG MFI) (sup. fig. 3f-k) . Plasma, IgA-plasma and IgA-/IgG-depleted plasma samples 509 with <70% IgA or IgG depletion in were all excluded from this study (sup. fig. 3b ). Only subjects 510 with all three plasma fractions (plasma, IgA-and IgA-/IgG depleted ) were included in final 511 analysis (n=30) (sup. fig. 3b ). 512 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.06.22270359 doi: medRxiv preprint 513 IgA spiking assay 514 Using RBDWT-ACE2 binding inhibition assay and the SARS-CoV-2 multiplex assay, purified IgA 515 was spiked back into IgA-plasma up to 100μg/ml of IgA (12.5, 50 and 100μg/ml) as proof of 516 concept that IgA contributes to ACE2 binding inhibition of convalescent plasma in a dose 517 dependent manor. 518 519 Fc effector functional assays 520 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 8, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 8, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 8, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 8, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.06.22270359 doi: medRxiv preprint Fc-mediated effector 650 function contributes to the in vivo antiviral effect of an HIV neutralizing antibody IgA is 655 Important for Clearance and Critical for Protection from Rotavirus Infection High-throughput, multiplexed IgG subclassing of antigen-specific antibodies 659 from clinical samples Distinct Features and Functions of Systemic and Mucosal Humoral Immunity Among 664 SARS-CoV-2 Convalescent Individuals Immunoglobulin isotype influences affinity and specificity The regulation of IgA class switching The Fc-mediated effector functions of a potent SARS-CoV-2 neutralizing 675 antibody, SC31, isolated from an early convalescent COVID-19 patient, are essential for 676 the optimal therapeutic efficacy of the antibody SARS-CoV-2 Variants of Concern Computational biophysical 681 characterization of the SARS-CoV-2 spike protein binding with the ACE2 receptor and 682 implications for infectivity Differential 687 Kinetics of Immune Responses Elicited by Covid-19 Vaccines Serum IgA Fc effector functions in 692 infectious disease and cancer A review 695 of COVID-19 convalescent plasma use in COVID-19 with focus on proof of efficacy Asymptomatic and symptomatic SARS-CoV-2 infections elicit polyfunctional antibodies Neutralizing antibody activity in convalescent sera from 707 infection in humans with SARS-CoV-2 and variants of concern A cross-reactive human IgA monoclonal antibody blocks SARS-CoV-2 713 spike-ACE2 interaction SARS-CoV-2 718 Neutralization with BNT162b2 Vaccine Dose 3 COVID-19-neutralizing antibodies predict disease severity and 726 survival Major role of IgM in the neutralizing activity of convalescent plasma 731 against SARS-CoV-2 Cytokine 734 profile and disease severity in patients with COVID-19 SARS-CoV-2 variants, spike mutations and 739 immune escape Variable Region 742 Identical Immunoglobulins Differing in Isotype Express Different Paratopes * Humoral and circulating follicular helper T cell responses in recovered 749 patients with COVID-19 Neutralizing antibody levels are 753 highly predictive of immune protection from symptomatic SARS-CoV-2 infection Role of IgM and IgA Antibodies in the Neutralization of SARS-CoV-2. 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