Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2
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Abstract
Isolation and characterization of antibodies in COVID-19 patients has largely focused on memory B cells, however it is the antibody-secreting plasma cells that are directly responsible for the production of serum antibodies, which play a critical role in controlling and resolving SARS-CoV-2 infection. To date there is little known about the specificity of plasma cells in COVID-19 patients. This is largely because plasma cells lack surface antibody expression, which complicates their screening. Here, we describe a technology pipeline that integrates single-cell antibody repertoire sequencing and high-throughput mammalian display screening to interrogate the specificity of plasma cells from 16 convalescent COVID-19 patients. Single-cell sequencing allows us to profile antibody repertoire features in these patients and identify highly expanded clonal lineages. Mammalian display screening is employed to reveal that 37 antibodies (out of 132 candidates) derived from expanded plasma cell clonal lineages are specific for SARS-CoV-2 antigens, including antibodies that target the receptor binding domain (RBD) with high affinity and exhibit potent neutralization of SARS-CoV-2.
One Sentence Summary
Single-cell antibody repertoire sequencing and high-throughput screening identifies highly expanded plasma cells from convalescent COVID-19 patients that produce SARS-CoV-2-specific antibodies capable of potent neutralization.
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SciScore for 10.1101/2021.02.12.430940: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethical statement: Ethics permission was granted by the ethics board “Ethikkommission Nordwest- und.
Consent: Every participant has signed a written informed consent as described previously (30).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 SARS-CoV-2 S1-, RBD-and S2-specific antibodies were evaluated individually by flow cytometry with mouse-Fc tagged antigens, followed by a secondary antibody tagged with Phycoerythrin (PE) or Allophycocyanin (APC). SARS-CoV-2suggested: NoneS1-suggested: NoneS2-specificsuggest…SciScore for 10.1101/2021.02.12.430940: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethical statement: Ethics permission was granted by the ethics board “Ethikkommission Nordwest- und.
Consent: Every participant has signed a written informed consent as described previously (30).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 SARS-CoV-2 S1-, RBD-and S2-specific antibodies were evaluated individually by flow cytometry with mouse-Fc tagged antigens, followed by a secondary antibody tagged with Phycoerythrin (PE) or Allophycocyanin (APC). SARS-CoV-2suggested: NoneS1-suggested: NoneS2-specificsuggested: Nonemouse-Fc tagged antigens,suggested: NonePhycoerythrin (PE)suggested: NoneAllophycocyanin (APCsuggested: NonePatients included in the subset were selected based on POCTs assessing the presence of IgG and IgM SARS-CoV-2-specific antibodies (Qingdao Hightop Biotech, #H100), performed at the time of blood collection, after resolution of symptoms as seen from Fig. 1. IgM SARS-CoV-2-specificsuggested: NoneExperimental Models: Cell Lines Sentences Resources Briefly, SARS-CoV-2 spike-pseudotyped lentiviruses incorporating a transfer plasmid encoding firefly luciferase were produced from HEK293T cells. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Pseudotyped virions standardized to an input producing ~100,000 RLUs were incubated with serial dilutions of recombinant antibodies for 60 min at 37 °C prior to the addition of ~15,000 HEK293T-ACE2 cells and incubation for 48h. HEK293T-ACE2suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Participants consisted of patients from the SERO-BL-COVID-19 study sponsored by the Department of Health, Canton Basel-Land, Switzerland. Canton Basel-Landsuggested: NoneSoftware and Algorithms Sentences Resources FACS was analyzed using FlowJo X software. FlowJosuggested: (FlowJo, RRID:SCR_008520)The raw FASTQ files from deep sequencing that support the findings of this study will be deposited (following peer-review and publication) in the Sequence Read Archive (SRA) with the primary accession code(s) . Sequence Read Archivesuggested: (DDBJ Sequence Read Archive, RRID:SCR_001370)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:While the linker-based design of the HDR construct improved and facilitated the generation of stable cell lines, reformatting does come with its limitations similar to known issues of converting antibody fragments (e.g., single-chain variable fragments and fragment antigen binding) to full-length IgG (56, 57), as well as affinity, stability and expression issues. Evaluating the RBD binders in the non-linker format increased the affinity and neutralizing potential, emphasizing the negative influence of a linker on antigen binding. This limitation may have caused a substantial loss in SARS-CoV-2 reactive sequences that do not tolerate the presence of the utilized 57-amino acid linker. Recently, Cao and colleagues have demonstrated how clonal expansion of Bmem can be used as a proxy for antigen specificity (13). However, despite finding two RBD-specific antibodies among the 132 candidates that were expressed, only one of them appeared to be (weakly) neutralizing, leading to the conclusion that antigen pre-selection was required. In this study, we found three potent neutralizing antibodies targeting the RBD from 132 selected clonal lineages, and while this is only a slight improvement in the success rate compared to Cao et al., it did demonstrate the potential to discover antibodies from PCs by single-cell sequencing. Given the unique physiology of PCs, this is particularly relevant at early stages of an infection as well as in the first weeks and months of a pandemic, when exped...
Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04383535 Completed Convalescent Plasma and Placebo for the Treatment of COVID-1… NCT04427501 Recruiting A Study of LY3819253 (LY-CoV555) and LY3832479 (LY-CoV016) i… NCT04452318 Recruiting COVID-19 Study Assessing the Efficacy and Safety of Anti-Spi… NCT04426695 Recruiting Safety, Tolerability, and Efficacy of Anti-Spike (S) SARS-Co… NCT04519437 Active, not recruiting Study Assessing the Safety, Tolerability, Pharmacokinetics, … 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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