Breadth and function of antibody response to acute SARS-CoV-2 infection in humans

  1. Kuan-Ying A. Huang
  2. Tiong Kit Tan
  3. Ting-Hua Chen
  4. Chung-Guei Huang
  5. Ruth Harvey
  6. Saira Hussain
  7. Cheng-Pin Chen
  8. Adam Harding
  9. Javier Gilbert-Jaramillo
  10. Xu Liu
  11. Michael Knight
  12. Lisa Schimanski
  13. Shin-Ru Shih
  14. Yi-Chun Lin
  15. Chien-Yu Cheng
  16. Shu-Hsing Cheng
  17. Yhu-Chering Huang
  18. Tzou-Yien Lin
  19. Jia-Tsrong Jan
  20. Che Ma
  21. William James
  22. Rodney S. Daniels
  23. John W. McCauley
  24. Pramila Rijal
  25. Alain R. Townsend

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Abstract

Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 13.0% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) and over half of anti-nucleocapsid (19 of 35) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-RBD, three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. At last, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.

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  1. Version published to 10.1101/2020.08.28.267526v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.08.28.267526: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: The study protocol and informed consent were approved by the ethics committee at the Chang Gung Medical Foundation and the Taoyuan General Hospital, Ministry of Health and Welfare.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Staining and sorting of plasmablasts: Freshly separated peripheral blood mononuclear cells (PBMCs) or thawed PBMCs were stained with fluorescent-labelled antibodies to cell surface markers purchased from BD Biosciences, USA; Pacific blue anti-CD3 (clone UCHT1, Cat. No. 558117, BD), Fluorescein isothiocyanate anti-CD19 (clone HIB19, Cat. No. 555412, BD), Phycoerythrin-Cy7 anti-CD27 (clone M-T271, Cat. No. 560609, BD), Allophycocyanin-H7 anti-CD20 (clone L27, Cat. No. 641396, BD), Phycoerythrin-Cy5 anti-CD38 (clone HIT2, Cat. No. 555461, BD) and Phycoerythrin anti-human IgG (clone G18-145, Cat. No. 555787, BD).
    anti-CD3
    suggested: (BD Biosciences Cat# 558117, RRID:AB_397038)
    anti-CD19
    suggested: (BD Biosciences Cat# 555412, RRID:AB_395812)
    anti-CD27
    suggested: (BD Biosciences Cat# 560609, RRID:AB_1727456)
    anti-CD20
    suggested: (BD Biosciences Cat# 641396, RRID:AB_1645724)
    anti-CD38
    suggested: (BD Biosciences Cat# 555461, RRID:AB_395854)
    Plates were washed and incubated with horseradish peroxidase-conjugated rabbit anti-human IgG secondary antibody (Rockland Immunochemicals, USA).
    anti-human IgG
    suggested: None
    Both MDCK-Spike and MDCK-RBD cells were then FACS sorted for highly expressing cells using the CR3022 antibody.
    CR3022
    suggested: None
    Bound primary antibodies were detected with FITC-conjugated anti-IgG secondary.
    anti-IgG
    suggested: None
    The anti-influenza human monoclonal antibody BS 1A, anti-SARS spike glycoprotein MAb CR3022 and convalescent serum were used as antibody controls for each experiment.
    anti-influenza
    suggested: None
    anti-SARS spike glycoprotein MAb CR3022
    suggested: None
    Plaque reduction neutralisation assay (Francis Crick Institute): Confluent monolayers of Vero E6 cells in 96-well plates were incubated with ~14 plaque forming units (PFU) of SARS CoV-2 (hCoV-19/England/02/2020, EPI_ISL_407073) and antibodies in a 2-fold dilution series (triplicates) for 3 hours at room temperature.
    EPI_ISL_407073
    suggested: None
    Antibody was biotinylated using EZ-Link Sulfo-NHS-LC-biotin (21237; Life Technologies) and then mixed with competing MAb (in at least 10-fold excess) and transferred to the blocked NUNC plates for 1 hour.
    Sulfo-NHS-LC-biotin
    suggested: None
    The control antibody (a non-blocking anti-influenza N1 MAb) or ACE2-Fc without antibody used to obtain the maximum signal and wells with PBS/BSA buffer only were used to determine the minimum signal.
    anti-influenza N1
    suggested: None
    50% inhibitory concentrations of the antibodies against ACE2 was determined using non-linear regression curve fit using GraphPad Prism 8.
    ACE2
    suggested: None
    After 1 hour a second layer Streptavidin-HRP antibody (S911, Life Technologies) diluted 1:1,600 in PBS/0.1% BSA (37525; Thermo Fisher Scientific) was added and incubated for another 1 hour.
    S911,
    suggested: (Imported from the IEDB Cat# S-9-11, RRID:AB_2848104)
    S911
    suggested: (Imported from the IEDB Cat# S-9-11, RRID:AB_2848104)
    Experimental Models: Cell Lines
    SentencesResources
    Plasmids were transfected into the HEK293T cell line for expression of recombinant full-length human IgG MAbs in serum-free transfection medium.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Flow-cytometry based binding assay: MDCK-Spike cells were produced by stably transfecting parental MDCK-SIAT1 cells with cDNA expressing full-length SARS-CoV-2 spike glycoprotein.
    MDCK-Spike
    suggested: None
    MDCK-RBD cells were produced by, stably transducing MDCK-SIAT1 cells with a Lentiviral vector encoding a cDNA expressing RBD amino acids 340-538 (NITN.
    MDCK-SIAT1
    suggested: ECACC Cat# 05071502, RRID:CVCL_Z936)
    MDCK-NTD cells were produced by stably transfecting parental MDCK-SIAT1 cells with cDNA expressing SARS-CoV-2 NTD.
    MDCK-NTD
    suggested: None
    MDCK-Spike or MDCK-RBD cells were prepared and resuspended.
    MDCK-RBD
    suggested: None
    Triplicate serial dilutions of antibody are pre-incubated with a fixed dose of SARS-CoV-2 (Australia/VIC01/2020, GenBank MT007544) (55) in triplicate before incubation with Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Briefly, Vero E6 cells were pre-seeded in a 96 well plate at a concentration of 104 cells per well.
    Vero E6
    suggested: None
    30 μl of biotinylated RBD (25 nM) were mixed and 50 μl of the mixture was then transferred to the MDCK-ACE2 cells.
    MDCK-ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    The binding activities were analyzed by BD FACSCanto™ II flow cytometer (BD Biosciences, USA)
    BD FACSCanto™
    suggested: None
    Data are analysed using four-parameter logistic regression (Hill equation) in GraphPad Prism 8.3.
    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: 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.

    About SciScore

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  3. Version published to 10.1101/2020.08.28.267526v1 on bioRxiv