PONE-D-21-26634IgG3 and IgM Identified as Key to SARS-CoV-2 Neutralization in Convalescent Plasma PoolsPLOS ONE

Dear Dr. Kalina

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. 

Two reviewers have assessed your manuscript. The reviewers consider your manuscript of interest, as addressing a problem of clinical importance. However, they expressed a series of concerns that need to be carefully addressed before we can consider your manuscript for publication. Therefore, we would like to invite you to REVISE your paper. In addition to the reviewer comments, I have also one concern. Sterlin et al [(Science translational medicine. 2021;13(577)] demonstrate that IgA dominates the early neutralizing antibody response to SARS-CoV-2. In your discussion, could you provide explanation to the differences between your and their findings.  

Please submit your revised manuscript by  the Nov 25 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. 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.

We look forward to receiving your revised manuscript.

Kind regards,

Gheyath K. Nasrallah

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Two reviewers have assessed your manuscript. The reviewers consider your manuscript of interest, as addressing a problem of clinical importance. However, they expressed a series of concerns that need to be carefully addressed before we can consider your manuscript for publication. Therefore, we would like to invite you to REVISE your paper. In addition to the reviewer comments, I have also one concern. Sterlin et al [(Science translational medicine. 2021;13(577)] demonstrate that IgA dominates the early neutralizing antibody response to SARS-CoV-2. In your discussion, could you provide explanation to the differences between your and their findings.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Competing Interests/Financial Disclosure* (delete as necessary) section:

“I have read the journal's policy and the authors of this manuscript have the following competing interests: All authors are employees of CSL Behring.”

We note that one or more of the authors are employed by a commercial company CSL Behring    1.    Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

    2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. 

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

3. Thank you for stating the following financial disclosure:

“The study was funded by CSL Behring GmbH.”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

4. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

   a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

   b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

5. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

6. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“The authors would like to acknowledge the following CSL employees for logistical support and laboratory analysis: Sara Stinca, Michèle Werren, Claudia Hadorn, Michaela Gerlach, Roland Weber, Harald Steller, Michelle Williams, Robin Jenness, Catherine Wright, Jessica Shan, Supavadee Amatayakul-Chantler.”

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“The study was funded by CSL Behring GmbH.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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: Yes

Reviewer #2: Yes

**********

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 manuscript in hand presents results on the potential role of IgM and IgG3, taken from convalescent plasma pools derived from individuals who recovered from the disease, to neutralize the virus in vitro.

The following remarks need your attention:

- What is the red line in Fig 1D? couldn’t delineate/differentiate colors in Fig. 1D!

- In page 4, the 1st paragraph speaks about ALL convalescent pools and how candidates were selected, and their plasma tested. In paragraph 2 of same page the authors discuss six pools in addition to the Australian pool. My assumption was that ALL pools were treated the same way in terms of having to wait for at least 28 days and not only the Australian pool, is this correct? - -

- The authors used pooled “donations”, were these donations similar to blood bank donations? If so, don’t you think that pooling 200-300 donations into one pool was too much? Such large pooling “system” has the potential to hide any Ig’s variation, why not use smaller number of donations (e.g. 20-50 donations) in each pool for more “realistic” approach? Also, What is the volume of each pool (i.e. volume range)? and how pooling was done?

- 1st and 2nd paragraphs of page 4 should be re-written for uniformity.

- Isn’t there a contradiction between Fig 1C, Fig 1D and Table 1 regarding IgM result outcomes? figure 1C shows background amounts of anti-S1 IgM antibodies which will not make a difference if depleted, whereas Table 1 speaks about significant contribution of IgM to the neutralization process (also line 200)!

- Lines 235-239: results of Fig 2 (A-C) show minimal or baseline amounts of IgM which contradicts what was exhibited in Fig 2D (specifically the IgM graph).

Reviewer #2: The manuscript IgG3 and IgM Identified as Key to SARS-CoV-2 Neutralization in Convalescent Plasma Pools may have important implications for the development of potent therapies for COVID-19. By analyzing plasma pools consisting of 247–567 individual convalescent donors, the authors demonstrated the binding of IgG3 and IgM to Spike-1 protein and the receptor-binding domain correlates strongly with viral neutralization in vitro. Furthermore, despite accounting for only approximately 12% of total immunoglobulin mass, collectively IgG3 and IgM account for approximately 80% of the total neutralization. The hyperimmune globulins with high IgG3 and IgM concentrations may be a highly efficacious therapy. My recommendations suggest minor revisions on this manuscript to be accepted in Plos One.

Some point should be addressed before resubmission:

1) Line 38 - Determine if there is an approval in the bioethics committee at this stage of the methodology. This was not commented in the manuscript.

2) Line 42 and others - In my way of understanding all the supplementary figures could be placed as the main ones. These figures contain a lot of relevant information fundamental to the consistency of the article.

3) Line 103 - to be more didactic about the presentation of antibody depletion, I find it interesting to put the scheme together with the methodology.

4) Line 142 – insert the reference of this methodology used by the group.

5) Line 155 - add reference too

6) Line 267 - Conversely, do the authors have any postulation or comment regarding the low efficiency of neutralization in IgG2 and IgG4?

**********

6. 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: Carlos Prudencio

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-21-26634R1IgG3 and IgM Identified as Key to SARS-CoV-2 Neutralization in Convalescent Plasma PoolsPLOS ONE

Dear Dr. Kalina

We are very close to acceptance. However, reviewer 2 has raised very important comment that needs to be address. I agree with his comment. To support your answer please refer to 

D Sterlin, A Mathian, M Miyara, et al. Iga dominates the early neutralizing antibody response to sars-cov-2. Science translational medicine 2021; 13(577).<o:p></o:p>

Please submit your revised manuscript by 16/12/2021. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. 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.

We look forward to receiving your revised manuscript.

Kind regards,

Gheyath K. Nasrallah

Academic Editor

PLOS ONE

Journal Requirements:

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.

[Note: HTML markup is below. Please do not edit.]

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 #1: (No Response)

Reviewer #2: All comments have been addressed

**********

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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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 #1: Yes

Reviewer #2: Yes

**********

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 #1: Yes

Reviewer #2: Yes

**********

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 #1: Since the last review, the authors have adequately addressed issues raised by the reviewer. My remaining issue is the IgA! In their rebuttal, the authors attached a table (Supplemental S2 Table: Anti-SARS-CoV-2 S1 antibody levels in smaller sized convalescent plasma pools consisting of 12-51 donors); the table clearly shows the dominance of IgA, as measured by AUC, as neutralizing Ab, although the authors still maintain that IgA is of less importance in this regard after 28 days! The pooled plasma used in the study is about 28 days post infection, and I understand that IgA was shown, in previous studies, of less importance and declines around a month post infection, your data says, in several places a different outcome! The question remains: Don't you think that IgA should still be considered as a valid option? The rationale for conducting the study is to justify, in the future, using IgG3 and IgM as passive therapeutic Abs. Don't you think that IgA would be a more valid candidate over IgG's and IgM especially in severe lung disease since it is a secretory Ab?

Reviewer #2: In this manuscript, the authors presented important informations for the development of potent therapies for COVID-19, as it indicates that hyperimmune globulins or convalescent plasma donations with high IgG3 concentrations may be a highly efficacious therapy. The authors have adequately addressed my comments and concerns raised.

**********

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 #1: No

Reviewer #2: Yes: Carlos Roberto Prudencio

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

IgG3 and IgM Identified as Key to SARS-CoV-2 Neutralization in Convalescent Plasma Pools

PONE-D-21-26634R2

Dear Dr. Kalina,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Gheyath K. Nasrallah

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: