Landscape analysis of escape variants identifies SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization
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Abstract
Although neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of COVID-19 vaccines and have received emergency use authorization as therapeutics, viral escape mutants could compromise their efficacy. To define the immune-selected mutational landscape in S protein, we used a VSV-eGFP-SARS-CoV-2-S chimeric virus and 19 neutralizing monoclonal antibodies (mAbs) against the receptor-binding domain (RBD) to generate 50 different escape mutants. The variants were mapped onto the RBD structure and evaluated for cross-resistance to mAbs and convalescent human sera. Each mAb had a unique resistance profile, although many shared residues within an epitope. Some variants ( e.g ., S477N) were resistant to neutralization by multiple mAbs, whereas others ( e.g ., E484K) escaped neutralization by convalescent sera, suggesting some humans induce a narrow repertoire of neutralizing antibodies. Comparing the antibody-mediated mutational landscape in S with sequence variation in circulating SARS-CoV-2, we define substitutions that may attenuate neutralizing immune responses in some humans.
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SciScore for 10.1101/2020.11.06.372037: (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. Table 2: Resources
Software and Algorithms Sentences Resources Non-linear regression (curve fit) was performed to calculate IC50 values for Fig 3C, 4B, S4, and S5A using Prism 8.0 (GraphPad). Prismsuggested: (PRISM, RRID:SCR_005375)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)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 …SciScore for 10.1101/2020.11.06.372037: (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. Table 2: Resources
Software and Algorithms Sentences Resources Non-linear regression (curve fit) was performed to calculate IC50 values for Fig 3C, 4B, S4, and S5A using Prism 8.0 (GraphPad). Prismsuggested: (PRISM, RRID:SCR_005375)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)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:Limitations of this study: Use of chimeric VSV that depends on SARS-CoV-2 S protein for entry into cells enabled the selection of 50 escape mutants. Although chimeric VSV serves as an effective mimic of SARS-CoV-2 S protein-mediated entry and viral neutralization, sequence analysis of circulating human isolates revealed that 34 of those escape mutants are present in the context of infectious SARS-CoV-2. The remaining 16 variants may represent S sequences with compromised fitness in the background of SARS-CoV-2 highlighting one potential limitation of our work. Additional limitations of our study are the relatively limited number of polyclonal human sera that we profiled against the panel of escape mutants. Additional human sera samples at lower dilutions may help determine the extent to which serum-based neutralization of virus is affected by individual or combinations of escape mutants.
Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04375046 Not yet recruiting Recombinant Bacterial ACE2 Receptors -Like Enzyme of B38-CAP… NCT04287686 Withdrawn Recombinant Human Angiotensin-converting Enzyme 2 (rhACE2) a… Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 42 and 44. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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 10.1101/2020.11.06.372037: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement The protocols were approved by the Institutional Animal Care and Use 503 Committee at the Washington University School of Medicine (Assurance number A3381-01). 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 2020b), and a separate study using three different antibodies defined resistance 270 substitutions at R346, N440, E484, F490 and Q493 (Weisblum et al., E484, F490suggested: NoneExperimental … SciScore for 10.1101/2020.11.06.372037: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement The protocols were approved by the Institutional Animal Care and Use 503 Committee at the Washington University School of Medicine (Assurance number A3381-01). 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 2020b), and a separate study using three different antibodies defined resistance 270 substitutions at R346, N440, E484, F490 and Q493 (Weisblum et al., E484, F490suggested: NoneExperimental Models: Cell Lines Sentences Resources MAbs in the left panel were purified from Expi293F cells transfected with antibody 437 expression vector (pABVec6W) expressing heavy chain V-D-J and light Chain V-J cloned from 438 single B cells. Expi293Fsuggested: RRID:CVCL_D615)444 Plaque assays were performed to validate the VSV-SARS-CoV-2 mutant on Vero cells in the 445 presence and absence of the mAb in the overlay. Verosuggested: NoneVero CCL81, Vero E6 and Vero E6-TMPRSS2 were maintained in DMEM 487 (Corning or VWR) supplemented with glucose, L-glutamine, sodium pyruvate, and 10% fetal 488 bovine serum (FBS). Vero CCL81suggested: NoneVero E6suggested: RRID:CVCL_XD71)Vero E6-TMPRSS2 cells were generated using a lentivirus vector described as previously 490 (Case et al., Vero E6-TMPRSS2suggested: NoneEscape clones were plaque-purified on Vero-E6 TMPRSS2 cells in the 495 presence of mAb, and plaques in agarose plugs were amplified on MA104 cells with the mAb 496 present in the medium. Vero-E6 TMPRSS2suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Experimental Models: Organisms/Strains Sentences Resources BALB/c mice were 523 immunized and boosted twice (two and four weeks later) with 5-10 µg of RBD and S protein (twice) sequentially, each adjuvanted with 50% AddaVax and give via an intramuscular route. BALB/csuggested: NoneSoftware and Algorithms Sentences Resources s. mAb neutralization assays using VSV-SARS-CoV-2were 548 conducted similarly but using an MOI of 100. 549 QUANTIFICATION AND STATISTICAL ANALYSIS 551 All statistical tests were performed using GraphPad Prism 8.0 software as described in 552 the indicated figure legends. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Non-linear regression (curve fit) was 554 performed for Fig 1A, S1A, S2A, and S7A using Prism 8.0. Prismsuggested: (PRISM, RRID:SCR_005375)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:
334 335 Limitations of this study 336 Use of chimeric VSV that depends on SARS-CoV-2 S protein for entry into cells greatly 337 facilitated the selection of 48 escape mutants. Although this chimeric VSV serves as an effective 338 mimic of SARS-CoV-2 spike mediated entry and viral neutralization, sequence analysis of 339 circulating human isolates reveals that 27 of those escape mutants are present in the context of 340 infectious SARS-CoV-2. The remaining 21 variants may represent S sequences with 341 compromised fitness in the background of SARS-CoV-2 highlighting one potential limitation of 342 our work. Additional limitations of our study are the relatively few polyclonal human sera that we 343 profiled against the panel of escape mutants that suggests substitutions at residue 484 are 344 associated with resistance. Additional human sera samples at lower dilution factors may help 345 determine the extent to which serum based neutralization of virus is affected by the escape 346 mutants. 347
Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04375046 Not yet recruiting Recombinant Bacterial ACE2 Receptors -Like Enzyme of B38-CAP... NCT04287686 Withdrawn Recombinant Human Angiotensin-converting Enzyme 2 (rhACE2) a... 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: Please consider improving the rainbow (“jet”) colormap used on pages 36 and 38. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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