Dear Dr. Guo,

Thank you for submitting your manuscript entitled "Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes" for consideration as a Research Article by PLOS Biology.

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Kind regards,

Paula

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Paula Jauregui, PhD,

Associate Editor

PLOS Biology

Dear Dr. Guo,

Thank you very much for submitting your manuscript "Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by several independent reviewers.

In light of the reviews (below), we are pleased to offer you the opportunity to address the comments from the reviewers in a revised version that we anticipate should not take you very long. We will then assess your revised manuscript and your response to the reviewers' comments and we may consult the reviewers again.

In particular, reviewer #1 says that you don’t provide sufficient experimental data to support the conclusion that the antibody binds to the RBD with “open” or “all close state”, and wants you to validate the immunofluorescence results using a published antibody. Please, also address the rest of the reviewers' concerns.

We expect to receive your revised manuscript within 1 month.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Paula

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Paula Jauregui, PhD,

Associate Editor,

pjaureguionieva@plos.org,

PLOS Biology

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REVIEWS:

Reviewer #1: Therapeutic antibodies.

Reviewer #2: Structure-based vaccine design and host immune response-pathogens interactions.

Reviewer #1: Major Points

1. The authors suggested monoclonal antibody PR1077 binds to the RBD of spike protein in either "open" or "all close state". The manuscript did not provide sufficient experimental data to support this conclusion. As shown in Fig S7, the binding epitope of PR1077 largely overlaps with hACE2 binding region. A CryEM structure of SARS-CoV-2 spike protein trimer has been recently published (Science 2020 367(6483):1260-1263). The authors should specify the critical binding epitopes of PR1077 exposed for surface binding in both "open" or "down" conformations (all close state).

2. Immunofluorescence staining assay was used instead of the classic PRNT assay. In this case, a reliable primer antibody for staining is critical to validate the assay. An in-house prepared and uncharacterized anti-SARS-CoV-2 rabbit serum sample was used to visualize virus infected cells. The authors should use other published neutralizing antibodies to confirm their results (Fig 2F).

3. Has PR1077 been tested against any new SARS-COV-2 variants? This would significantly increase the impact of this story.

Minor points:

1. Several COVID-19 vaccines have been already approved. The statement "Currently, there is no approved vaccines or therapeutics against COVID-19" in the abstract needs to be revised.

2. A statistical test needs to be added in Fig 5B and C for comparison between the PR1077 treated group and PBS control.

3. More discussion of PR961 neutralization mechanism could be added. PR961 appeared to be a non-receptor blocking neutralizing antibody, which is unique as compared to other published neutralizing antibodies.

Reviewer #2: Fu et al. reported the use of a transgenic mouse platform to generate antibodies against the RBD of the SARS-CoV-2 spike protein. The authors identified 25 potently neutralizing RBD-specific antibodies and determined the structure of three antibodies with novel epitopes on the RBD. They further showed the prophylactic or therapeutic efficacy of the PR1077 antibody against SARS-CoV-2 infection. In addition, the binding and neutralization ability of PR1077 against numerous emerging RBD mutants was demonstrated. I recommend acceptance with minor revisions.

Minor revisions:

1) In paragraph 3 of the "Selection and characterization of the purified antibodies" section, the authors claimed that the VH3-33 germline gene usage is among the most frequently used but do not include a citation. Recent reviews do not show this germline gene among the most used but do show the similar VH3-30 gene among the top 5-6. The similarity of the germline genes was later mentioned in the Discussion section when the comparison to published VH3-30 antibody structures was discussed.

2) Figure 1D is never cited in the manuscript.

Dear Dr. Guo,

Thank you for submitting your revised Research Article entitled "Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes" for publication in PLOS Biology. I have now obtained advice from reviewer #1 and have discussed their comments with the Academic Editor. 

Based on the reviews, we will probably accept this manuscript for publication, provided you satisfactorily address the following data and other policy-related requests.

DATA POLICY:

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797 

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

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We expect to receive your revised manuscript within two weeks.

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Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

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*Early Version*

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*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please do not hesitate to contact me should you have any questions.

Sincerely,

Paula

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Paula Jauregui, PhD,

Associate Editor,

pjaureguionieva@plos.org,

PLOS Biology

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Reviewer remarks:

Reviewer #1: The revised version addressed reviewer's concerns. It is recommended for acceptance.

Dear Dr. Guo,

On behalf of my colleagues and the Academic Editor, Ken Cadwell, I am pleased to say that we can in principle offer to publish your Research Article "Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes" in PLOS Biology, provided you address any remaining formatting and reporting issues.

Please ensure that figure legends in your manuscript include information on where the underlying data can be found.

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Thank you again for supporting Open Access publishing. We look forward to publishing your paper in PLOS Biology. 

Sincerely, 

Paula

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Paula Jauregui, PhD 

Associate Editor 

PLOS Biology