Identification of neutralizing human monoclonal antibodies from Italian Covid-19 convalescent patients

  1. Emanuele Andreano
  2. Emanuele Nicastri
  3. Ida Paciello
  4. Piero Pileri
  5. Noemi Manganaro
  6. Giulia Piccini
  7. Alessandro Manenti
  8. Elisa Pantano
  9. Anna Kabanova
  10. Marco Troisi
  11. Fabiola Vacca
  12. Dario Cardamone
  13. Concetta De Santi
  14. Chiara Agrati
  15. Maria Rosaria Capobianchi
  16. Concetta Castilletti
  17. Arianna Emiliozzi
  18. Massimiliano Fabbiani
  19. Francesca Montagnani
  20. Emanuele Montomoli
  21. Claudia Sala
  22. Giuseppe Ippolito
  23. Rino Rappuoli

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Abstract

In the absence of approved drugs or vaccines, there is a pressing need to develop tools for therapy and prevention of Covid-19. Human monoclonal antibodies have very good probability of being safe and effective tools for therapy and prevention of SARS-CoV-2 infection and disease. Here we describe the screening of PBMCs from seven people who survived Covid-19 infection to isolate human monoclonal antibodies against SARS-CoV-2. Over 1,100 memory B cells were single-cell sorted using the stabilized prefusion form of the spike protein and incubated for two weeks to allow natural production of antibodies. Supernatants from each cell were tested by ELISA for spike protein binding, and positive antibodies were further tested for neutralization of spike binding to receptor(s) on Vero E6 cells and for virus neutralization in vitro. From the 1,167 memory B specific for SARS-CoV-2, we recovered 318 B lymphocytes expressing human monoclonals recognizing the spike protein and 74 of these were able to inhibit the binding of the spike protein to the receptor. Finally, 17 mAbs were able to neutralize the virus when assessed for neutralization in vitro . Lead candidates to progress into the drug development pipeline will be selected from the panel of neutralizing antibodies identified with the procedure described in this study.

One Sentence Summary

Neutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for therapeutic and prophylactic interventions.

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  1. ScreenIT

    SciScore for 10.1101/2020.05.05.078154: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: IRCCS – Lazzaro Spallanzani Rome (IT) and Azienda Ospedaliera Universitaria Senese, Siena (IT) that provided samples from SARS-CoV-2 convalescent donors who gave their written consent.
    IRB: The study was approved by local ethics committees (Parere 18_2020 in Rome and Parere 17065 in Siena) and conducted according to good clinical practice in accordance with the declaration of Helsinki (European Council 2001, US Code of Federal Regulations, ICH 1997).
    RandomizationThis study was unblinded and not randomized.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Blood samples were screened for SARS-CoV-2 RNA and for antibodies against HIV, HBV and HCV.
    antibodies against HIV
    suggested: None
    HCV
    suggested: None
    ELISA assay with S1 and S2 subunits of SARS-CoV-2 S-protein: The presence of S1- and S2-binding antibodies in culture supernatants of monoclonal S-protein-specific memory B cells was assessed by means of an ELISA assay implemented with the use of a commercial kit (ELISA Starter Accessory Kit, Catalogue No. E101; Bethyl Laboratories, Montgomery, TX, USA).
    S2-binding
    suggested: None
    After an incubation of 1 h at 37°C, plates were washed and incubated with 25 μl/well secondary antibody (horseradish peroxidase (HRP)-conjugated goat anti-human IgG-Fc Fragment polyclonal antibody, diluted 1:10,000 in blocking buffer, Catalogue No. A80-104P; (Bethyl Laboratories, Montgomery, TX, USA) for 1 h at 37°C.
    anti-human IgG-Fc
    suggested: (Bethyl Cat# A80-104P, RRID:AB_67064)
    25 μL/well of alkaline phosphatase-conjugated goat anti-human IgG (Sigma-Aldrich) and IgA (Jackson Immuno Research) were used as secondary antibodies.
    alkaline phosphatase-conjugated goat anti-human IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 109-055-190, RRID:AB_2888997)
    anti-human IgG
    suggested: None
    PNPP (p-nitrophenyl phosphate) (Thermo Fisher) was used as soluble substrate to detect SARS-CoV-2 S-protein specific monoclonal antibodies and the final reaction was measured by using the Varioskan Lux Reader (Thermo Fisher Scientific) at a wavelength of 405 nm.
    ( p-nitrophenyl phosphate )
    suggested: None
    p-nitrophenyl phosphate
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    For virus propagation, sub-confluent Vero E6 cell monolayers were prepared in T175 flasks (Sarstedt) containing supplemented D-MEM high glucose medium.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    The protein was purified from filtered cell supernatants using NiNTA resin (GE Healtcare #11-0004-58)
    GE Healtcare
    suggested: None
    Data were analyzed using the FlowJo data analysis software package (TreeStar, USA).
    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.
    • 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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  2. Version published to 10.1101/2020.05.05.078154v2 on bioRxiv
  3. Version published to 10.1101/2020.05.05.078154v1 on bioRxiv