Heterologous SARS-CoV-2 IgA neutralising antibody responses in convalescent plasma

  1. Samantha K Davis
  2. Kevin John Selva
  3. Ester Lopez
  4. Ebene R Haycroft
  5. Wen Shi Lee
  6. Adam K Wheatley
  7. Jennifer A Juno
  8. Amy Adair
  9. Phillip Pymm
  10. Samuel J Redmond
  11. Nicholas A Gherardin
  12. Dale I Godfrey
  13. Wai-Hong Tham
  14. Stephen J Kent
  15. Amy W Chung

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Abstract

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.

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  1. ScreenIT

    SciScore for 10.1101/2022.02.06.22270359: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsField Sample Permit: Ethics statement: The study protocols were approved by the University of Melbourne Human Research Ethics Committee (#2056689) and all associated procedures were carried out in accordance with the approved guidelines.
    IRB: Ethics statement: The study protocols were approved by the University of Melbourne Human Research Ethics Committee (#2056689) and all associated procedures were carried out in accordance with the approved guidelines.
    Consent: All participants provided written informed consent in accordance with the Declaration of Helsinki.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Briefly, anti-IgG or IgA capture antibody was coated on Maxisorb 96 well plates (Nunc) overnight at 4°C.
    anti-IgG
    suggested: None
    SARS-CoV-2 bead-based multiplex assay: The SARS-CoV-2 specific antibody isotypes (IgM, IgG and IgA1) were assessed using a multiplex assay as previously described (Selva et al., 2021).
    IgM , IgG
    suggested: None
    IgA1
    suggested: None
    Phycoerythrin (PE)-conjugated mouse anti-human pan-IgG and IgA1 (Southern Biotech) (1.3μg/ml) were added to detect SARS-CoV-2 specific antibodies.
    anti-human pan-IgG
    suggested: None
    Titrations of pooled convalescent plasma and an anti-SARS-CoV-2 RBD neutralising human IgG1 antibody (SAD-S35, ACRO Biosystems, USA) were included as positive controls and uninfected subject plasma was included as negative controls.
    anti-SARS-CoV-2
    suggested: None
    Briefly, an array of SARS-CoV-2 antigens including S1 (Sino Biological), RBD wild-type (WT) (B; wild-type, Wuhan) and 18 RBD single mutants used in figure 3a with 5 additional RBD mutants were included in figure 3b-e, in a bead suspension containing 700 beads per bead region were added to each well (20ul), with biotinylated Avitag-ACE2 (kindly provided by Dale Godfrey, Nicholas Gherardin and Samuel Redmond) at a final concentration of 12.5μg/ml per well, and dilutions of plasma or purified antibodies were added to 384-well plates.
    antigens including S1
    suggested: None
    RBD neutralising human IgG1 antibody (SAD-S35, ACRO Biosystems, USA) was included as a positive control, in addition to COVID-19 negative plasma and buffer only negative controls.
    human IgG1
    suggested: (Abcam Cat# ab20402, RRID:AB_445563)
    Matching dilutions to account for loss of antibody during depletion process: Anti-RBDWT IgM (sup. fig. 3c) and IgG (sup. fig. 3d) binding was also assessed for plasma, IgA- and IgA-/IgG-depleted plasma via multiplex.
    depletion process: Anti-RBDWT IgM ( sup . fig . 3c )
    suggested: None
    IgG ( sup . fig . 3d ) binding
    suggested: None
    Quality control testing of antibody depleted plasma: Successful depletion IgG and IgA was confirmed for matched dilutions via IgG SARS-CoV-2 RBDWT multiplex (sup. fig. 3f-k, sup. table 2).
    3f-k , sup . table 2
    suggested: None
    Data normalisation: For purified antibody binding to the RBD single mutants of subjects with IgA neutralisation (n=13), antibody MFI values were normalised to the maximum antibody binding (100%) (IgG and IgA) to each variant to account for differences in coupling efficiency for the RBD mutants.
    n=13
    suggested: (Advanced Targeting Systems Cat# BT-N13, RRID:AB_10972554)
    IgA
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    THP-1 and Ramos S-orange cell association assay: A THP-1 and Ramos S-orange cell association was used as previously described (W. S. Lee et al., 2021).
    THP-1
    suggested: None
    Ramos
    suggested: None
    Opsonised Ramos S-orange cells and THP-1 monocytes were incubated for 1 hour under cell culture conditions before fixation.
    Ramos S-orange
    suggested: None
    Software and Algorithms
    SentencesResources
    Cells were acquired by flow cytometry using the BD LSR Fortessa with a high-throughput sampler attachment (HTS) and the data was analysed using FlowJo 10.7.1 (sup. fig. 6b for gating strategy).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Data analysis: Traditional statistical analyses were performed with Graphpad Prism 9.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    ACE2 binding inhibition) using Matlab with the statistics and machine learning toolbox (Mathsworks) and PLS_Toolbox (Eigenvector).
    Matlab
    suggested: (MATLAB, RRID:SCR_001622)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2022.02.06.22270359 on medRxiv
  3. Version published to 10.1002/cti2.1424