High-resolution epitope mapping and characterization of SARS-CoV-2 antibodies in large cohorts of subjects with COVID-19
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Abstract
As Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread, characterization of its antibody epitopes, emerging strains, related coronaviruses, and even the human proteome in naturally infected patients can guide the development of effective vaccines and therapies. Since traditional epitope identification tools are dependent upon pre-defined peptide sequences, they are not readily adaptable to diverse viral proteomes. The Serum Epitope Repertoire Analysis (SERA) platform leverages a high diversity random bacterial display library to identify proteome-independent epitope binding specificities which are then analyzed in the context of organisms of interest. When evaluating immune response in the context of SARS-CoV-2, we identify dominant epitope regions and motifs which demonstrate potential to classify mild from severe disease and relate to neutralization activity. We highlight SARS-CoV-2 epitopes that are cross-reactive with other coronaviruses and demonstrate decreased epitope signal for mutant SARS-CoV-2 strains. Collectively, the evolution of SARS-CoV-2 mutants towards reduced antibody response highlight the importance of data-driven development of the vaccines and therapies to treat COVID-19.
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SciScore for 10.1101/2020.11.23.20235002: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Participation in the study was voluntary and the study protocol was approved by the Yale Institutional Review Board. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: The cell line was obtained from the ATCC and has been tested negative for contamination with mycoplasma. Table 2: Resources
Antibodies Sentences Resources Plates were washed, then incubated with HRP-goat anti-human IgG or HRP-donkey anti-human IgM (Jackson ImmunoResearch) secondary antibody diluted 1/10,000 in blocking buffer for 1 hour at room temperature. anti-human IgGsuggested: Noneanti-human …SciScore for 10.1101/2020.11.23.20235002: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Participation in the study was voluntary and the study protocol was approved by the Yale Institutional Review Board. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: The cell line was obtained from the ATCC and has been tested negative for contamination with mycoplasma. Table 2: Resources
Antibodies Sentences Resources Plates were washed, then incubated with HRP-goat anti-human IgG or HRP-donkey anti-human IgM (Jackson ImmunoResearch) secondary antibody diluted 1/10,000 in blocking buffer for 1 hour at room temperature. anti-human IgGsuggested: Noneanti-human IgMsuggested: NoneDevelopment of a diagnostic classifier for COVID-19: To generate a diagnostic score that classified subjects as serologically positive for antibodies to COVID-19, motif enrichment values were normalized using the mean and standard deviation of enrichments within the training set of pre-pandemic control repertoires. COVID-19suggested: NoneExperimental Models: Cell Lines Sentences Resources SARS-CoV-2, strain USA-WA1/2020, was obtained from BEI Resources (#NR-52281) and was amplified in VeroE6 cells. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Software and Algorithms Sentences Resources The surface of the protein structure was calculated using the MSMS program62 with a probe radius of 2.5 Å. MSMSsuggested: (MSMS, RRID:SCR_003532)A multiple sequence alignment of all these coronavirus sequences was performed using Clustal Omega72. Clustal Omega72suggested: 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: We detected the following sentences addressing limitations in the study:We acknowledge various limitations with the SERA platform that impact this study. Much of this study has focused on dominant epitopes prevalent in COVID-19 cases, but many of the private epitopes not explicitly discussed here, particularly in spike RBD, are critical to fully understanding the protective antibody response and clinical outcomes. Moreover, there are clear limitations for probing the epitope repertoire with linear peptides, chiefly the challenges of identifying structural epitopes and the role of post-translational modifications such as glycans56. While a random peptide library enables unique opportunities to identify structural mimics, much work remains in cataloguing and mapping these mimics to their cognate antigens. In summary, we present the application of SERA to assess SARS-CoV-2 seropositivity and to characterize a high-resolution map of motifs and epitopes in individuals and populations. We demonstrate the ability of the platform to assess disease severity, to identify structural motifs associated with neutralization, to compare in silico epitope response to multiple coronavirus strains, to assess potential immune escape at sites of variation, to evaluate longitudinal changes in signal, and to reveal potential autoantigen response, all with one assay. The random nature of the libraries, the ability to identify linear mimics of structural epitopes, and the ability to leverage quality-controlled reference data from a large pre-pandemic cohort all contribut...
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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SciScore for 10.1101/2020.11.23.20235002: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Participation in the study was voluntary and the study protocol was approved by the Yale Institutional Review Randomization To assess the significance of this decreased epitope signal, we in silico randomly mutated amino acids throughout the same protein sequences as a null distribution. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication The cell line was obtained from the ATCC and has been tested negative for contamination with mycoplasma. Table 2: Resources
Antibodies Sentences Resources Antibody- bound bacterial clones were selected with 50 µL Protein A/G Sera-Mag SpeedBeads (GE Life Sciences, … SciScore for 10.1101/2020.11.23.20235002: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Participation in the study was voluntary and the study protocol was approved by the Yale Institutional Review Randomization To assess the significance of this decreased epitope signal, we in silico randomly mutated amino acids throughout the same protein sequences as a null distribution. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication The cell line was obtained from the ATCC and has been tested negative for contamination with mycoplasma. Table 2: Resources
Antibodies Sentences Resources Antibody- bound bacterial clones were selected with 50 µL Protein A/G Sera-Mag SpeedBeads (GE Life Sciences, cat#17152104010350) (IgG) or by incubation with a biotinylated anti-human IgM antibody (Jackson ImmunoResearch, cat# 709-066-073) final assay dilution 1:100, followed by a second incubation with 50 ul Dynabead MyOne anti-human IgMsuggested: (Jackson ImmunoResearch Labs Cat# 709-066-073, RRID:AB_2340508)Plates were washed, then incubated with HRP- goat anti-human IgG or HRP-donkey anti-human IgM (Jackson ImmunoResearch) secondary antibody diluted 1/10,000 in blocking buffer for 1 hour at room temperature. anti-human IgGsuggested: NoneDevelopment of a diagnostic classifier for COVID-19 To generate a diagnostic score that classified subjects as serologically positive for antibodies to COVID-19, motif enrichment values were normalized using the mean and standard deviation of enrichments within the training set of pre-pandemic control repertoires. COVID-19suggested: NoneExperimental Models: Cell Lines Sentences Resources The mixture was subsequently incubated with VeroE6 cells in a 6-well plate for 1hour, for adsorption. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Software and Algorithms Sentences Resources The surface of the protein structure was calculated using the MSMS program62 with a probe radius of 2.5 Å. MSMSsuggested: (MSMS, RRID:SCR_003532)A multiple sequence alignment of all these coronavirus sequences was performed using Clustal Omega72. Clustal Omega72suggested: 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: We detected the following sentences addressing limitations in the study:
We acknowledge various limitations with the SERA platform that impact this study. Much of this study has focused on dominant epitopes prevalent in COVID-19 cases, but many of the private epitopes not explicitly discussed here, particularly in spike RBD, are critical to fully understanding the protective antibody response and clinical outcomes. Moreover, there are clear limitations for probing the epitope repertoire with linear peptides, chiefly the challenges of identifying structural epitopes and the role of post-translational modifications such as glycans56. While a random peptide library enables unique opportunities to identify structural mimics, much work remains in cataloguing and mapping these mimics to their cognate antigens. In summary, we present the application of SERA to assess SARS-CoV-2 seropositivity and to characterize a high-resolution map of motifs and epitopes in individuals and populations. We demonstrate the ability of the platform to assess disease severity, to identify structural motifs associated with neutralization, to compare in silico epitope response to multiple coronavirus strains, to assess potential immune escape at sites of variation, to evaluate longitudinal changes in signal, and to reveal potential autoantigen response, all with one assay. The random nature of the libraries, the ability to identify linear mimics of structural epitopes, and the ability to leverage quality-controlled reference data from a large pre-pandemic cohort all contribut...
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.
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