Cross-neutralization antibodies against SARS-CoV-2 and RBD mutations from convalescent patient antibody libraries
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Abstract
The emergence of coronavirus disease 2019 (COVID-19) pandemic led to an urgent need to develop therapeutic interventions. Among them, neutralizing antibodies play crucial roles for preventing viral infections and contribute to resolution of infection. Here, we describe the generation of antibody libraries from 17 different COVID-19 recovered patients and screening of neutralizing antibodies to SARS-CoV-2. After 3 rounds of panning, 456 positive phage clones were obtained with high affinity to RBD (receptor binding domain). Then the positive clones were sequenced and reconstituted into whole human IgG for epitope binning assays. After that, all 19 IgG were classified into 6 different epitope groups or Bins. Although all these antibodies were shown to have ability to bind RBD, the antibodies in Bin2 have more superiority to inhibit the interaction between spike protein and angiotensin converting enzyme 2 receptor (ACE2). Most importantly, the antibodies from Bin2 can also strongly bind with mutant RBDs (W463R, R408I, N354D, V367F and N354D/D364Y) derived from SARS-CoV-2 strain with increased infectivity, suggesting the great potential of these antibodies in preventing infection of SARS-CoV-2 and its mutations. Furthermore, these neutralizing antibodies strongly restrict the binding of RBD to hACE2 overexpressed 293T cells. Consistently, these antibodies effectively neutralized pseudovirus entry into hACE2 overexpressed 293T cells. In Vero-E6 cells, these antibodies can even block the entry of live SARS-CoV-2 into cells at only 12.5 nM. These results suggest that these neutralizing human antibodies from the patient-derived antibody libraries have the potential to become therapeutic agents against SARS-CoV-2 and its mutants in this global pandemic.
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SciScore for 10.1101/2020.06.06.137513: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. 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 Anti-RBD antibody measurement: 18 plasma samples from COVID-19 patients and 3 plasma samples from healthy donors were enrolled in this study. Anti-RBDsuggested: NoneWells were washed three times with PBST (0.1% Tween 20 in PBS) followed by the addition of HRP-conjugated antibody against human IgG and subsequent incubation for 1 hour at room temperature. human IgGsuggested: NoneAfter three washes with PBST, the bound antibodies were detected by … SciScore for 10.1101/2020.06.06.137513: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. 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 Anti-RBD antibody measurement: 18 plasma samples from COVID-19 patients and 3 plasma samples from healthy donors were enrolled in this study. Anti-RBDsuggested: NoneWells were washed three times with PBST (0.1% Tween 20 in PBS) followed by the addition of HRP-conjugated antibody against human IgG and subsequent incubation for 1 hour at room temperature. human IgGsuggested: NoneAfter three washes with PBST, the bound antibodies were detected by HRP-conjugated anti-Myc antibody followed by incubation with TMD. anti-Mycsuggested: NoneAfter three washes with PBST, the bound antibodies were detected by HRP-conjugated anti-human Fc antibody followed by incubation with TMD and the color reaction was measured. anti-human Fcsuggested: NoneThe mixture was added to 293T-ACE2 cells, incubated at 4°C for 1 hour, washed 3 times with PBS before APC-conjugated anti-mouse Fc antibody was added. anti-mousesuggested: NoneEpitope binning experiments of anti-SARS-CoV-2 Antibodies were performed in 96-channel mode with in tandem format. anti-SARS-CoV-2suggested: NoneCells were immunolabelled for 1.5 hours at room temperature with the rabbit anti SARS-CoV-2 spike antibody, washed three times with PBS, and followed by the addition of Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody. anti SARS-CoV-2suggested: Noneanti-rabbit IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources The mixture was added to 293T-ACE2 cells, incubated at 4°C for 1 hour, washed 3 times with PBS before APC-conjugated anti-mouse Fc antibody was added. 293T-ACE2suggested: RRID:CVCL_YZ65)Briefly, 293T cells were transfected with psPAX2 and vectors encoding SARS-CoV-2 spike protein as well as a core plasmid expressing RFP. 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)For immunofluorescence (IF) analysis, Vero-E6 cells were transfected and fixed on day 6 post-transfection in 80% acetone for 10 min at room temperature. Vero-E6suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Neutralization activity of mAbs against live SARS-CoV-2: SARS-CoV-2 obtained from a sputum sample was amplified in Vero-E6 to make working stocks of the virus. SARS-CoV-2suggested: NoneResults 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 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.
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