Induction of cross-reactive antibody responses against the RBD domain of the spike protein of SARS-CoV-2 by commensal microbiota

  1. Justus Ninnemann
  2. Lisa Budzinski
  3. Marina Bondareva
  4. Mario Witkowski
  5. Stefan Angermair
  6. Jakob Kreye
  7. Pawel Durek
  8. S. Momsen Reincke
  9. Elisa Sánchez-Sendin
  10. Selin Yilmaz
  11. Toni Sempert
  12. Gitta Anne Heinz
  13. Caroline Tizian
  14. Martin Raftery
  15. Günther Schönrich
  16. Daria Matyushkina
  17. Ivan V. Smirnov
  18. Vadim M. Govorun
  19. Eva Schrezenmeier
  20. Thomas Dörner
  21. Silvia Zocche
  22. Edoardo Viviano
  23. Katharina Johanna Sehmsdorf
  24. Hyun-Dong Chang
  25. Philipp Enghard
  26. Sascha Treskatsch
  27. Andreas Radbruch
  28. Andreas Diefenbach
  29. Harald Prüss
  30. Mir-Farzin Mashreghi
  31. Andrey A. Kruglov

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Abstract

The commensal microflora is a source for multiple antigens that may induce cross-reactive antibodies against host proteins and pathogens. However, whether commensal bacteria can induce cross-reactive antibodies against SARS-CoV-2 remains unknown. Here we report that several commensal bacteria contribute to the generation of cross-reactive IgA antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. We identified SARS-CoV-2 unexposed individuals with RBD-binding IgA antibodies at their mucosal surfaces. Conversely, neutralising monoclonal anti-RBD antibodies recognised distinct commensal bacterial species. Some of these bacteria, such as Streptococcus salivarius , induced a cross-reactive anti-RBD antibodies upon supplementation in mice. Conversely, severely ill COVID-19 patients showed reduction of Streptococcus and Veillonella in their oropharynx and feces and a reduction of anti-RBD IgA at mucosal surfaces. Altogether, distinct microbial species of the human microbiota can induce secretory IgA antibodies cross-reactive for the RBD of SARS-CoV-2.

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  1. Version published to 10.1101/2021.08.08.455272v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.08.08.455272: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Human Donors: The recruitment of study subjects was conducted in accordance with the Ethics Committee of the Charité (EA 1/144/13 with EA 1/075/19, EA 2/066/20) and was in compliance with the Declaration of Helsinki.
    Field Sample Permit: Bacteria culture: PYG medium and plates were prepared as described by the DSMZ (German Collection of Microorganisms and Cell Cultures). 300,000 events were sorted into 1 ml of PYG medium and directly transferred to a COY anaerobic chamber.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Staining for human immunoglobulins was performed in 100 µL with 1:50 (v/v) of the detection antibodies: anti-human IgM Brilliant Violet 650 (clone: MHM-88, Biolegend® Cat. No. 314526)
    anti-human IgM Brilliant Violet 650
    suggested: None
    For the detection of Spike protein-similar structures the samples were first incubated in 50 µL containing 0.5 µg SARS-CoV-2 Spike Neutralizing Antibody (clone: HA14JL2302, Sino Biological Inc. Cat. No: 40592-R001) or Neutralizing Antibody isolated from COVID-19 patients for 15 min at 4 °C then washed with PBS and stained again in 50 µL of the anti-Rabbit Alexa 647 (7,5µg/ml, Jackson ImmunoResearch Cat. No. 111-606-144) or anti-human IgG PE/ Dazzle™ 594 (2µg/ml) which was then topped up with 5 µM Hoechst 33342 solution
    anti-Rabbit
    suggested: (Jackson ImmunoResearch Labs Cat# 111-606-144, RRID:AB_2338083)
    anti-human IgG
    suggested: None
    Enzyme-linked immunosorbent assay: For the detection of antibody titers in sera and fecal supernatants 96-well plates were coated with goat anti-human Ig (H+L chain) antibody (Southern Biotech, Cat. No. 2010-01) or goat anti-human IgA Fab (Southern Biotech, Cat. No. 2050-01) antibody for the detection of IgG, IgM and IgA respectively.
    anti-human Ig ( H+L chain )
    suggested: None
    anti-human IgA
    suggested: (SouthernBiotech Cat# 2050-01, RRID:AB_2795701)
    After that, plates were washed 5 times with 200 μL of 1x PBST and detection antibodies were applied: anti-human IgG-AP (ICN/Cappel, Cat No. 59289),
    anti-human IgG-AP
    suggested: None
    Microarray was incubated with monoclonal anti-RBD antibodies (final concentration 1 mcg/ml) or mouse fecal supernatants (1:1 dilution) at 30 C for 1 hours with constant rotation.
    anti-RBD
    suggested: None
    , anti-human IgG-Alexa647 (Southern Biotech; Cat. No.: 2040-31), goat anti-Mouse IgA Antibody DyLight® 650 (Bethyl Laboratories; Cat.No.: A90-103D5) at 30 C for 1 hour.
    anti-human IgG-Alexa647
    suggested: None
    anti-Mouse IgA
    suggested: (Bethyl Cat# A90-103D5, RRID:AB_10630982)
    Experimental Models: Cell Lines
    SentencesResources
    Flow cytometric assay for analysis of ACE2-RBD interaction: HEK293T cells were transfected with a plasmid expressing human ACE2 protein.
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Experimental Models: Organisms/Strains
    SentencesResources
    C57Bl/6 mice were injected with 200 µl of heat-killed bacteria i.p. From oral gavage, live bacteria stocks were grown, washed with PBS several times, OD600 was adjusted to 1, 200 µl of live bacteria was gavaged every second day.
    C57Bl/6
    suggested: None
    Recombinant DNA
    SentencesResources
    Protein expression: Uncharacterised protein RSSL-01370 was amplified from the genomic DNA of Streptococcus salivarius K12 using the following primers: 5’s-CTCCATATGAATTTACCAAGTCACCATACAAGGG -’s3 and 5’-GTGGTCGACATTCACTTTTTCAGTTGCTACACC -’3 and subsequently cloned into pET-21b containing NdeI and XhoI restriction sites.
    pET-21b
    suggested: RRID:Addgene_132607)
    Software and Algorithms
    SentencesResources
    Raw data were processed and de-multiplexed using MiSeq Reporter Software.
    MiSeq
    suggested: (A5-miseq, RRID:SCR_012148)
    Forward and reverse reads were combined using PANDAseq 2.11 with a minimum overlap of 25 bases (PMID:22333067) and classified using “classifier.jar” 2.13 from the Ribosomal Database Project with a confidence cutoff of 50% (PMID: 24288368, PMID: 17586664).
    PANDAseq
    suggested: (PANDAseq, RRID:SCR_002705)
    The copy number adjusted counts were agglomerated to bacterial genera, rarefied to the smallest size and alpha diversity were estimated using phyloSeq 1.34 (PMID: 23630581).
    phyloSeq
    suggested: (phyloseq, RRID:SCR_013080)
    The linear discriminant analysis were performed using LEfSe, based on copy number adjusted counts normalized to 1M reads [46].
    LEfSe
    suggested: (LEfSe, RRID:SCR_014609)
    Raw sequence data were deposited at the NCBI Sequence Read Archive (SRA) under the accession number PRJNA738291.
    NCBI Sequence Read Archive
    suggested: (NCBI Sequence Read Archive (SRA, RRID:SCR_004891)
    Sequence identity was determined with the Nucleotide Basic Local Alignment Search Tool (BLAST) provided by NCBI.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    Spike glycoprotein (SPIKE) wild type + mutations (JPT Peptide Technologies GmbH; RT-MW-WCPV-S-V02)
    SPIKE
    suggested: (SPIKE, RRID:SCR_010466)
    , anti-human IgG-Alexa647 (Southern Biotech; Cat. No.: 2040-31), goat anti-Mouse IgA Antibody DyLight® 650 (Bethyl Laboratories; Cat.No.: A90-103D5) at 30 C for 1 hour.
    Bethyl Laboratories
    suggested: (Bethyl, RRID:SCR_013554)
    Samples were acquired on a FACSCanto (BD Biosciences) and analyzed using FlowJo v10
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Correspondence of the found MS/MS fragments to the proteins was performed with the help of Biotools software (Bruker Daltonik, Germany) and a Mascot MS/MS ion search.
    Biotools
    suggested: None

    Results from OddPub: Thank you for sharing your data.

    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.

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  3. Version published to 10.1101/2021.08.08.455272v1 on bioRxiv