Dear Prof. Mandelboim,

Thank you very much for submitting your manuscript "SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, 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.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

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

Sincerely,

Kanta Subbarao

Section Editor

PLOS Pathogens

Kanta Subbarao

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors have explored the very well-known interaction between the SARS-CoV-2 RBD and human ACE2 and the potential to block this interaction, demonstrating what others have previously shown – that soluble dimeric forms of these proteins can inhibit SARS-CoV-2 infection vitro and in vivo. The activity they observe is clear, but relatively inefficient and does not compare well with many of the highly potent antibody therapies that achieve the same goal. Indeed, a dose of 75ug RBD-dimer per mouse was only partially effective at preventing infection (50% survival of mice at this dose) and the authors did not explore higher doses to see if prevention of infection was even possible with these reagents.

The observation that the RBD was more effective than the soluble ACE2 molecule was interesting and a possible explanation is provided (that increased virus proliferation outpaces the ACE-2 dimer reagent), but the evidence to support this explanation was very limited and other potential explanations also exist that were not explored, for example reagent stability; and ability of the reagent to bind to bind via both arms of the dimer may be easier for RBD dimer binding to cellular ACE2 than ACE2 dimer binding to virus RBD. Furthermore, if the reagent worked well in the first place, then there should not be increased virus proliferation. Regardless, the observations in this study seem incremental over what is already known.

Reviewer #2: The manuscript from Chaout and Achdout describes the role of SARS-CoV-2 RBD or ACE2 proteins fused to Ig domain as a possible treatment against SARS-CoV-2. The authors characterize both these fusion proteins for binding via flow cytometry to either Spike or ACE2-expressing cell lines, in plaque reduction neutralization assays (PRNT) and in in vivo humanized ACE2 mouse model. They also try to understand the mechanisms of inhibition by RBD-Ig or ACE2-Ig to determine why RBD-Ig is better at neutralization compared to ACE2-Ig but unfortunately are unable to firmly determine the precise mechanism. The manuscript could be improved by providing more controls, average values with errors rather than one representative value (example) or determining if differential affinities between the RBD-Ig and ACE2-Ig is a reason for the higher neutralization potency of the former.

1. Figure 1. In general, the manuscript defaults in showing one representative result without presenting the average values and errors from repeat experiments. In addition, it would be appropriate to include more controls such as non-specific spike expression, isotype controls or a different human receptor.

a. For Figure 1a-c, please include results from the other two representative experiments in terms of a column graph showing MFIs. This would provide an ability to show reproducibility between repeats.

b. For 1a, is there another cell line expressing a non-relevant human receptor in addition to 293-T ACE2 that could be used that would show that RBD-Ig would be specific to 293T-ACE2? Can the authors please add an isotype control to show that the staining is specific to anti-Flag and not to secondary antibody?

c. For 1b, can the authors use another cell line expressing mouse ACE2 or another non-relevant human receptor to show that RBD-Ig is specific to 293T-ACE2? Also would be good to use an isotype control or a RBD to another coronavirus that does not bind to 293T-ACE2?

d. For 1d, please see comments for 1a and 1b.

2. For Figure 2c and 2d, can the authors please provide the PRNT IC50 values for all three experiments in terms of nM (vs ug/ml). This would allow the comparison of the neutralization potency between ACE2-Ig and RBD-Ig.

3. For Fig. 3, is it possible to also show the viral load data in terms of TCID50 or RT-qPCR to complement the weight loss data? Or any histopathological data?

4. One major possibility to explain the increased potency of RBD-Ig vs ACE2-Ig is that RBD-Ig has higher binding affinity to ACE2 compared to ACE2-Ig binding to RBD.

a. Can the authors provide any evidence of this using biophysical assays such as surface plasmon resonance or bio-layer interferometry?

b. If affinity is a potential mechanism, this could be formally tested by using a RBD variant with the 501Y mutation which shows increased affinity to human/mouse ACE2 and could be a very powerful experiment to show development of an improved therapeutic.

5. Discussion: In general the Discussion is lacking in direct and more thorough comparisons between previously published work (Huang et al, EMBO and Iwanaga et al). A deeper comparison would be helpful to understand the differences in potency or in vivo protection.

6. While these are ACE2 decoys, a discussion of Linsky et al, Science 2020 and Tanaka et al, bioarchive 2021 (doi: https://doi.org/10.1101/2021.03.09.434641) would also be appropriate in the Discussion. In particular, if there are insights to what drives neutralization mechanisms.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: Please refer to the section above with relation to comment 1 to 4.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

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 #1: No

Reviewer #2: Yes: Wai-Hong Tham

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that 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 to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Prof. Mandelboim,

Thank you very much for submitting your manuscript "SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please address the comments from both of the reviewers.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, 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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

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

Sincerely,

Kanta Subbarao

Section Editor

PLOS Pathogens

Kanta Subbarao

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Please address the comments from both of the reviewers.

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors state that they are unaware of other studies that have used RBD-Fc to block in vivo. They are correct in that RBD-Fc may not have been used to block infection in vivo, prior to their study, but soluble RBD protein has been used, in Syrian hamster model (Zharadnik et al 2021, Nature Microbiology) - (previously published in BioRXiv in January 2021). Admittedly this was a higher affinity RBD, but on the other hand, it was a monomer, not a dimer which would limit its affinity. This is still a relevant point in the context of the current study. Furthermore, another paper published this year has also shown that RBD-Fc can inhibit infection in vivo (Shin et al 2021. Int.J.Biol.Sci). These two studies are not cited in the current manuscript and they reduce the novelty of this paper.

I agree with the authors that a soluble RBD reagent may in theory have a better chance of preventing escape from variants, compared to some therapeutic antibodies, - this is a good idea!. However, my concern is that in this study, this RBD-Ig tool does not appear to be a very effective therapeutic in the first place, at least in the mouse model that has been used. It showed a moderate improvement in mouse survival (50% surviving rather than 20%) and a moderate ~50% reduction in PFU/lung. For these reason, I do not see that this tool is likely to be an improvement over the many different monoclonal antibody therapeutics that are in use, or in development, some of which are effective against a range of variants. Unfortunately this reduces the impact of this study.

Reviewer #2: The authors have answered all my queries and modified the text.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: The authors have removed results from all of Figure 1. As such, Figure 1 is just a summary image, perhaps useful to just consolidate Figure 1 and 2 together?

**********

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 #1: No

Reviewer #2: Yes: Wai-Hong Tham

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that 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 to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Prof. Mandelboim,

We are pleased to inform you that your manuscript 'SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kanta Subbarao

Section Editor

PLOS Pathogens

Kanta Subbarao

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Thank you for addressing the issues raised by the reviewers.

Reviewer Comments (if any, and for reference):