PONE-D-23-12939Discovery of neutralizing SARS-CoV-2 antibodies highly enriched in a unique antigen specific B cell clusterPLOS ONE

Dear Dr. Tolstrup,

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Additional Editor Comments:

I suggest the authors to go through all the comments from reviewers and address them in the revised version of the manuscript

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study by the Tolstrup lab describes the discovery and characterizati on of neutralizing SARS-CoV-2

anti bodies . This is a very complete study with a clear and comprehendible experimental design

protocol, convincing and complete data and conclusions supported by well controlled study. The

manuscript can be published as submitt ed

Reviewer #2: The manuscript by Lende et al is comprehensive and detailed, with good observations and analysis of data obtained using several sophisticated techniques. The approach was to classify B cells from convalescent patients into transcriptomic profile groups. Single-cell sequence information was used to generate monoclonal antibodies via cloning, transfection and expression in ExpiCho-S cells. Resulting monoclonal antibodies were then screened for antigen binding, neutralisation activity against SARS-CoV-2 pseudoviruses and authentic viruses including major variants of concern. Finally, highly neutralising or broadly neutralising monoclonal antibodies were tested for in vivo protection against disease in a mouse model, validating the predictive value of the transcriptomic profiling approach.

The manuscript is generally well written, in clear standard English but will need thorough proof-reading to correct e.g. spacing errors.

Requested clarifications:

In the abstract and throughout the main body of the text, the step from characterising B cells to screening mAbs is skipped over. Only in the methods section is the generation of mAbs from the sequence data even mentioned. It would greatly assist readers’ understanding the concept of the approach used if the mAb generation was made clearer when describing the study.

Detailed characterisation was carried out on antibody 29044, but this was ‘identified in a pilot run’ (line 150). Were transcriptomic profiling and screening carried out on this pilot run? If they were, can the information be compiled into that for the main run? If they were not, then including this antibody as an exemplar of the profiling approach as an effective pre-screening method is inappropriate and misleading.

Have the authors considered population or demographic differences between studies as potentially contributing to the differences identified in lines 91-94?

Why were switched isotypes IgG2, IgG3 and IgG4 excluded (line 125), as these were highly mutated from the germline?

Line 154: “The vast majority of neutralizing mAbs were RBD binders” however Figure 4A shows that most neutralising mAbs have weaker binding to the RBD than to the overall Spike.

Please include results from the in vivo dose finding studies.

Line 316: So what timepoint of blood sampling is recommended for mAb discovery? Was there any attempt to correlate neutralising antibody identification with time of PBMC sampling? Table 1 describes the time from diagnosis to inclusion ie PBMC sampling, but would time from symptom onset be a better indicator for participant groups 2 & 3?

Minor comments

Line 95: needs to refer to Fig 2E as well as 2D.

Line 111: CD80 is not shown in Fig 2

Lines 162-164: add comment on BA.1

Line 172: There is no blue in Fig 5A. The legend and Fig 5A describe Groups A-C, and text describes Groups 1-4. Please make text consistent with figure.

Line 205: Gamma is not included in Fig 6D-F.

Line 206: Fig 6D-F shows BA.2, not BA.5

Line 314: “mAb” in both instances on this line is probably actually referring to “antibodies”, not monoclonals.

Resolution & image quality of figures, especially Fig 2, could be better.

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: PLOSOne-Tolstrup.pdf

Discovery of neutralizing SARS-CoV-2 antibodies highly enriched in a unique antigen specific B cell cluster

PONE-D-23-12939R1

Dear Dr. Tolstrup,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Nagarajan Raju

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: (No Response)

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: (No Response)

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: (No Response)

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

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