Expansion of SARS-CoV-2–Specific Antibody-Secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients

  1. Renata Varnaitė
  2. Marina García
  3. Hedvig Glans
  4. Kimia T. Maleki
  5. John Tyler Sandberg
  6. Janne Tynell
  7. Wanda Christ
  8. Nina Lagerqvist
  9. Hilmir Asgeirsson
  10. Hans-Gustaf Ljunggren
  11. Gustaf Ahlén
  12. Lars Frelin
  13. Matti Sällberg
  14. Kim Blom
  15. Jonas Klingström
  16. Sara Gredmark-Russ

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Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and has since become a global pandemic. Pathogen-specific Abs are typically a major predictor of protective immunity, yet human B cell and Ab responses during COVID-19 are not fully understood. In this study, we analyzed Ab-secreting cell and Ab responses in 20 hospitalized COVID-19 patients. The patients exhibited typical symptoms of COVID-19 and presented with reduced lymphocyte numbers and increased T cell and B cell activation. Importantly, we detected an expansion of SARS-CoV-2 nucleocapsid protein–specific Ab-secreting cells in all 20 COVID-19 patients using a multicolor FluoroSpot Assay. Out of the 20 patients, 16 had developed SARS-CoV-2–neutralizing Abs by the time of inclusion in the study. SARS-CoV-2–specific IgA, IgG, and IgM Ab levels positively correlated with SARS-CoV-2–neutralizing Ab titers, suggesting that SARS-CoV-2–specific Ab levels may reflect the titers of neutralizing Abs in COVID-19 patients during the acute phase of infection. Last, we showed that IL-6 and C-reactive protein serum concentrations were higher in patients who were hospitalized for longer, supporting the recent observations that IL-6 and C-reactive protein could be used as markers for COVID-19 severity. Altogether, this study constitutes a detailed description of clinical and immunological parameters in 20 COVID-19 patients, with a focus on B cell and Ab responses, and describes tools to study immune responses to SARS-CoV-2 infection and vaccination.

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  1. Version published to 10.4049/jimmunol.2000717
  2. Version published to 10.1101/2020.05.28.118729v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.05.28.118729: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Ethics statement: The study was approved by the Regional Ethical Review Board in Stockholm, Sweden and by the Swedish Ethical Review Authority.
    Consent: All COVID-19 patients and healthy controls included in this study provided a written informed consent for participation.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableStudy subjects and sampling of peripheral blood: Peripheral blood samples were collected from 20 adult COVID-19 patients hospitalized in April 2020 at the Karolinska University Hospital in Stockholm, Sweden (5 females and 15 males; age range between 34 and 67 years; median age 53 years) (Table I).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The following monoclonal antibody conjugates were used for cell surface staining: anti-CD8-Qdot605 (3B5) (Thermo Fisher Scientific), anti-CD19-BUV395 (SJ25C1), anti-CD14-V500 (MφP9), anti-CD4 -BUV737 (RPA-T4) (all from BD Biosciences), anti-CD123-BV510 (6H6), anti-CD27-BV650 (O323), anti-CD20-FITC (2H7), anti-CD38-BV421 (HB-7), anti-IgD-PE-Cy7 (IA6-2), anti-IgM-BV785 (MHM-88) (all from BioLegend), anti-CD3-PE-Cy5 (UCHT1), anti-CD56-ECD (N901) (all from Beckman Coulter), and anti-IgA-APC (REA1014) (Miltenyi).
    anti-CD8-Qdot605
    suggested: None
    anti-CD19-BUV395
    suggested: None
    anti-CD14-V500
    suggested: None
    anti-CD4
    suggested: None
    anti-CD123-BV510
    suggested: None
    anti-CD27-BV650
    suggested: None
    anti-CD20-FITC
    suggested: (MBL International Cat# K0170-4, RRID:AB_590955)
    anti-CD38-BV421
    suggested: None
    anti-IgD-PE-Cy7
    suggested: None
    anti-IgM-BV785
    suggested: None
    anti-CD3-PE-Cy5
    suggested: None
    anti-CD56-ECD (N901
    suggested: None
    anti-IgA-APC (REA1014
    suggested: None
    The following monoclonal antibody conjugates were used for intracellular staining: anti-IgG-PE (HP6017) (BioLegend) and anti-Ki-67-AF700 (B56) (BD Biosciences).
    anti-IgG-PE
    suggested: (Miltenyi Biotec Cat# 130-099-201, RRID:AB_2661448)
    HP6017
    suggested: None
    anti-Ki-67-AF700
    suggested: None
    B56
    suggested: None
    FluoroSpot assay for antibody-secreting cells: The number of SARS-CoV-2 nucleocapsid (N) protein-specific IgA, IgG and IgM antibody-secreting cells (ASCs), as well as the total number of IgA-, IgG- and IgM-ASCs in freshly isolated PBMCs were measured using a multicolor B cell FluoroSpot kit with modifications (Mabtech).
    IgA, IgG
    suggested: None
    Briefly, ethanol-activated IPFL membrane plates were coated overnight with either: (i) anti-IgG, anti-IgA, and anti-IgM capture antibodies (15µg/mL of each) for the detection of all ASCs, or (ii) SARS-CoV-2 N protein (10 µg/mL) for the detection of SARS-CoV-2-specific ASCs.
    anti-IgG
    suggested: None
    anti-IgM
    suggested: None
    Plates were then incubated at 37°C in 5% CO2 for 20 hours and then developed with anti-human IgG-550 (yellow fluorescence), anti-human IgA-490 (green fluorescence) and anti-human IgM-640 (red fluorescence) secondary detection antibodies (diluted 1:500 each) (all antibodies from Mabtech).
    anti-human IgG-550
    suggested: None
    anti-human IgA-490
    suggested: None
    anti-human IgM-640
    suggested: None
    For analysis of total SARS-CoV-2 IgG antibody titers, serum samples were serially diluted from 1:20 to 1:5120. 25 µL of diluted serum was then added to fixed cells and incubated at 37°C for 30 min, after which the slides were washed in NaCl for 30 min.
    SARS-CoV-2 IgG
    suggested: None
    Bound SARS-CoV-2 IgG antibodies were then detected by incubating for 30 min at 37°C with a secondary AF488-conjugated AffiniPure goat anti-human IgG antibody (Jackson Immunoresearch), diluted 1:200 in 0.1% Evan’s Blue.
    Bound SARS-CoV-2 IgG antibodies
    suggested: None
    anti-human IgG
    suggested: None
    ELISAs: SARS-CoV-2 specific IgG and IgA antibodies in serum were detected using anti-SARS-CoV-2 ELISA kits (both from Euroimmun), according to the manufacturer’s instructions.
    IgA
    suggested: None
    anti-SARS-CoV-2
    suggested: None
    SARS-CoV-2 specific IgM antibodies were detected using EDI Novel Coronavirus COVID-19 IgM ELISA kit (Epitope Diagnostics), according to the manufacturer’s instructions.
    specific IgM
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Immunofluorescence assay (IFA) for IgG against SARS-CoV-2: Vero E6 cells were infected with SARS-CoV-2 (isolate SARS-CoV-2/human/SWE/01/2020, accession number MT093571) for 24 hours, trypsinized and mixed with uninfected Vero E6 cells, and then seeded on microscope slides.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Finally, samples were incubated in a 2% formaldehyde solution (Polysciences) for 2 h, washed and resuspended in flow cytometry buffer, and data subsequently acquired on a BD LSRFortessa flow cytometer equipped with 355, 405, 488, 561, and 639 nm lasers and BD FACSDiva Software (BD Biosciences).
    BD FACSDiva
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    The following monoclonal antibody conjugates were used for cell surface staining: anti-CD8-Qdot605 (3B5) (Thermo Fisher Scientific), anti-CD19-BUV395 (SJ25C1), anti-CD14-V500 (MφP9), anti-CD4 -BUV737 (RPA-T4) (all from BD Biosciences), anti-CD123-BV510 (6H6), anti-CD27-BV650 (O323), anti-CD20-FITC (2H7), anti-CD38-BV421 (HB-7), anti-IgD-PE-Cy7 (IA6-2), anti-IgM-BV785 (MHM-88) (all from BioLegend), anti-CD3-PE-Cy5 (UCHT1), anti-CD56-ECD (N901) (all from Beckman Coulter), and anti-IgA-APC (REA1014) (Miltenyi).
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    Statistics and data analysis: Statistical analyses were performed using GraphPad Prism software 7.0 for MacOSX (GraphPad Software).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    FlowJo software version 10.5.3 (Tree Star) was used to analyze all flow cytometry data.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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  4. Version published to 10.1101/2020.05.28.118729v1 on bioRxiv