Persisting Neutralizing Activity to SARS-CoV-2 over Months in Sera of COVID-19 Patients

  1. Bertram Flehmig
  2. Michael Schindler
  3. Natalia Ruetalo
  4. Ramona Businger
  5. Manfred Bayer
  6. Angelika Haage
  7. Thomas Kirchner
  8. Karin Klingel
  9. Andrea Normann
  10. Lutz Pridzun
  11. Despina Tougianidou
  12. Michael B. Ranke

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Abstract

The relationship between the nasopharyngeal virus load, IgA and IgG antibodies to both the S1-RBD-protein and the N-protein, as well as the neutralizing activity (NAbs) against SARS-CoV-2 in the blood of moderately afflicted COVID-19 patients, needs further longitudinal investigation. Several new serological methods to examine these parameters were developed, validated and applied in three patients of a family which underwent an ambulatory course of COVID-19 for six months. The virus load had almost completely disappeared after about four weeks. Serum IgA levels to the S1-RBD-protein and, to a lesser extent, to the N-protein, peaked about three weeks after clinical disease onset but declined soon thereafter. IgG levels rose continuously, reaching a plateau at approximately six weeks, and stayed elevated over the observation period. Virus-neutralizing activity reached a peak about 4 weeks after disease onset but dropped slowly. The longitudinal associations of virus neutralization and the serological immune response suggest immunity in patients even after a mild clinical course of COVID-19.

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  1. Version published to 10.3390/v12121357
  2. ScreenIT

    SciScore for 10.1101/2020.08.20.20174912: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Written informed consents were obtained from all blood donors prior to the study.
    IRB: The study was approved by the Independent Research Ethics Committee (IEC) of the University of Tuebingen, IEC-Project Number 672/2020A Patients: All patients are related and live in the same household.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablePatient 1: Adult male.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Detection of IgA and IgG Antibodies against SARS-CoV-2-S1-RBD-Protein: IgG Antibody Assay: The ELISA test system E 111-IVD developed by Mediagnost GmbH, Reutlingen (Germany) was employed according to the manufacturer’s instructions.
    SARS-CoV-2-S1-RBD-Protein: IgG
    suggested: None
    This test system is a two-step enzyme-linked immunosorbent assay for the detection of IgG antibodies directed against SARS-CoV-2 S1, specifically targeting the receptor binding domain (RBD) protein.
    SARS-CoV-2
    suggested: None
    S1
    suggested: None
    Next, a horseradish peroxidase (HRP) conjugated goat anti-human IgG binds to the human IgG antibodies.
    anti-human IgG
    suggested: None
    Increasing extinctions represent increasing amounts of antibodies to SARS-CoV-2 S1 protein.
    SARS-CoV-2 S1 protein .
    suggested: None
    IgA Antibody Assay: The ELISA test system for the detection of IgA antibodies to SARS-CoV-2 S1-RBD protein was analogous to the anti-SARS-CoV-2 IgG ELISA, except that the HRP-labeled detection antibody was directed against human IgA antibodies.
    anti-SARS-CoV-2 IgG
    suggested: None
    human IgA
    suggested: None
    IgA and IgG assays for the determination of antibodies to the SARS-CoV-2 nucleoprotein (N-protein) in sera were developed in analogy to the assays for anti-SARS-CoV-2-S1-RBD-protein as described above.
    IgA
    suggested: None
    SARS-CoV-2 nucleoprotein ( N-protein
    suggested: None
    anti-SARS-CoV-2-S1-RBD-protein
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Virus Neutralization Assay (VNA): By means of the VNA, we quantified in vitro the ability of human sera from patients to inhibit the infection of human cells Caco-2 (human colorectal adenocarcinoma) with a strain of SARS-CoV-2 virus.
    Caco-2
    suggested: None
    To generate icSARS-CoV-2-mNG stocks, Caco2 cells were infected, the supernatant was harvested 48 hours post infection, centrifuged and stored at -80°C.
    Caco2
    suggested: None
    Software and Algorithms
    SentencesResources
    In order to quantify the infection rates, images were taken with the Cytation3 (Biotek Hoechst) and mNG+ cells were automatically counted by the Gen5 Software (Biotek).
    Gen5
    suggested: (Gen5, RRID:SCR_017317)
    Titers of neutralizing antibodies (NAbs) were calculated as the half-maximal inhibitory dose (ID50) using 4-parameter nonlinear regression (GraphPad Prism).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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.
    • Thank you for including a protocol registration statement.

    About SciScore

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  3. Version published to 10.1101/2020.08.20.20174912v1 on medRxiv