Identification of neutralizing human monoclonal antibodies from Italian Covid-19 convalescent patients
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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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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 Statement Consent: 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).Randomization This study was unblinded and not randomized. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
… 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 Statement Consent: 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).Randomization This study was unblinded and not randomized. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Blood samples were screened for SARS-CoV-2 RNA and for antibodies against HIV, HBV and HCV. antibodies against HIVsuggested: NoneHCVsuggested: NoneELISA 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-bindingsuggested: NoneAfter 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-Fcsuggested: (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 IgGsuggested: (Jackson ImmunoResearch Labs Cat# 109-055-190, RRID:AB_2888997)anti-human IgGsuggested: NonePNPP (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: Nonep-nitrophenyl phosphatesuggested: NoneExperimental Models: Cell Lines Sentences Resources For virus propagation, sub-confluent Vero E6 cell monolayers were prepared in T175 flasks (Sarstedt) containing supplemented D-MEM high glucose medium. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources The protein was purified from filtered cell supernatants using NiNTA resin (GE Healtcare #11-0004-58) GE Healtcaresuggested: NoneData were analyzed using the FlowJo data analysis software package (TreeStar, USA). FlowJosuggested: (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.
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