Kinetics and Isotype Assessment of Antibodies Targeting the Spike Protein Receptor Binding Domain of SARS-CoV-2 In COVID-19 Patients as a function of Age and Biological Sex

  1. Nancy R. Graham
  2. Annalis N. Whitaker
  3. Camilla A. Strother
  4. Ashley K. Miles
  5. Dore Grier
  6. Benjamin D. McElvany
  7. Emily A. Bruce
  8. Matthew E. Poynter
  9. Kristen K. Pierce
  10. Beth D. Kirkpatrick
  11. Renee D. Stapleton
  12. Gary An
  13. Jason W. Botten
  14. Jessica W. Crothers
  15. Sean A. Diehl

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Abstract

SARS-CoV-2 is the newly emerged virus responsible for the global COVID-19 pandemic. There is an incomplete understanding of the host humoral immune response to SARS-CoV-2 during acute infection. Host factors such as age and sex as well the kinetics and functionality of antibody responses are important factors to consider as vaccine development proceeds. The receptor-binding domain of the CoV spike (RBD-S) protein is important in host cell recognition and infection and antibodies targeting this domain are often neutralizing. In a cross-sectional study of anti-RBD-S antibodies in COVID-19 patients we found equivalent levels in male and female patients and no age-related deficiencies even out to 93 years of age. The anti-RBD-S response was evident as little as 6 days after onset of symptoms and for at least 5 weeks after symptom onset. Anti-RBD-S IgG, IgM, and IgA responses were simultaneously induced within 10 days after onset, but isotype-specific kinetics differed such that anti-RBD-S IgG was most sustained over a 5-week period. The kinetics and magnitude of neutralizing antibody formation strongly correlated with that seen for anti-RBD-S antibodies. Our results suggest age- and sex-related disparities in COVID-19 fatalities are not explained by anti-RBD-S responses. The multi-isotype anti-RBD-S response induced by live virus infection could serve as a potential marker by which to monitor vaccine-induced responses.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.15.20154443: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: University of Vermont Institutional Review Board approval was granted under registration STUDY00881.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Spike Glycoprotein Receptor Binding Domain (RBD) from SARS-CoV-2, Wuhan-Hu-1, was also used as a positive control during assay set up and this reagent was produced in HEK293T cells under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH: Spike Glycoprotein Receptor Binding Domain (RBD) from SARS-Related Coronavirus 2, Wuhan-Hu-1, Recombinant from NR-52306. Preparation of CR3022 monoclonal antibody: CR3022 is a SARS-CoV S-specific antibody originally isolated by single chain variable region phage display and then cloned as an IgG1/kappa monoclonal human IgG1/κ (13).
    NIH: Spike Glycoprotein Receptor Binding Domain (RBD
    suggested: None
    Cells were permeabilized with 0.1% 100X Triton in 1X PBS for 15 minutes and then incubated with a primary, cross-reactive rabbit anti-SARS-CoV N monoclonal antibody (40143-R001, Sinobiological) (1:20,000) followed by a peroxidase-labeled goat anti-rabbit antibody (5220-0336, SeraCare) (1:2,000) and then the peroxidase substrate (5510-0030, SeraCare).
    anti-SARS-CoV N
    suggested: None
    anti-rabbit
    suggested: (SeraCare KPL Cat# 5220-0336, RRID:AB_2857917)
    Experimental Models: Cell Lines
    SentencesResources
    Spike Glycoprotein Receptor Binding Domain (RBD) from SARS-CoV-2, Wuhan-Hu-1, was also used as a positive control during assay set up and this reagent was produced in HEK293T cells under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH: Spike Glycoprotein Receptor Binding Domain (RBD) from SARS-Related Coronavirus 2, Wuhan-Hu-1, Recombinant from NR-52306. Preparation of CR3022 monoclonal antibody: CR3022 is a SARS-CoV S-specific antibody originally isolated by single chain variable region phage display and then cloned as an IgG1/kappa monoclonal human IgG1/κ (13).
    HEK293T
    suggested: None
    Recombinant CR3022 was expressed in 293A cells (Invitrogen) by polyethyleneimine (Polysciences Inc.) transfection of 9 µg each of CR3022-HC and LC, culture for 7 days, and protein A agarose bead purification as described (34).
    293A
    suggested: None
    Vero E6 cells were maintained in complete Dulbecco’s Modified Eagle Medium (cDMEM) (11965–092) containing 10% fetal bovine serum (FBS) (16140–071), 1% HEPES Buffer Solution (15630–130), and 1% penicillin-streptomycin (15140–122) purchased from Thermo Fisher Scientific (Carlsbad, CA).
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    spike area under the curve (AUC) was calculated in Prism 8.4.3 (Graphpad Inc) from the OD405nm values from all six dilutions and using the negative control cutoff values as the baseline.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)
    FRNT50 determinations were made using a non-linear regression curve fit (log[inhibitor] vs. normalized response – variable slope) in GraphPad Prism. Graphics and Statistical testing: All statistics and graphics were performed using R version 3.6.1 using standard packages or GraphPad Prism 8.4.3.
    GraphPad Prism
    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: We detected the following sentences addressing limitations in the study:
    A limitation of our study is that we only followed symptomatic patients admitted to hospital; it is unclear whether antibody responses differ in asymptomatic or mildly symptomatic patients. We also did not directly assess whether the RBD-specific antibodies we studied were neutralizing at the clonal level, though we did observe a strong association with polyclonal RBD-S IgG responses and SARS-CoV-2 neutralizing activity. This is in agreement with other reports which confirm that RBD-S IgG levels correlate with neutralizing activity and that the RBD of SARS-CoV-2 is a potent target for neutralizing antibodies (16–18, 20, 21, 33). It will be important to determine whether anti-RBD IgA or even IgM antibodies contribute to blocking activity.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

    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 10.1101/2020.07.15.20154443v1 on medRxiv
  3. Version published to 10.1002/cti2.1189