PPATHOGENS-D-25-01596

Antigen flexibility supports the avidity of hemagglutinin-specific antibodies at low antigen densities

PLOS Pathogens

Dear Dr. Vahey,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens'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.

Please submit your revised manuscript within 30 days Nov 29 2025 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 plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ 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 editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Alan G. Goodman

Academic Editor

PLOS Pathogens

Thomas Hoenen

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

Your revised manuscript has been positively reviewed by the three reviewers. Reviewer #1 has remaining minor concerns that should be addressed before moving forward with your manuscript. In Figure S3, please highlight the data points in cluster "i" that correspond to the stem antibodies. Additionally, the dataset should be included as a supplementary table listing each antibody with its most frequent inter-HA spacing and angle. The Github link needs correcting too.

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

1) We ask that a manuscript source file is provided at Revision. Please upload your manuscript file as a .doc, .docx, .rtf or .tex. If you are providing a .tex file, please upload it under the item type u2018LaTeX Source Fileu2019 and leave your .pdf version as the item type u2018Manuscriptu2019.

2) Please provide an Author Summary. This should appear in your manuscript between the Abstract (if applicable) and the Introduction, and should be 150-200 words long. The aim should be to make your findings accessible to a wide audience that includes both scientists and non-scientists. Sample summaries can be found on our website under Submission Guidelines:

https://journals.plos.org/plospathogens/s/submission-guidelines#loc-parts-of-a-submission

3) Please upload all main figures as separate Figure files in .tif or .eps format. For more information about how to convert and format your figure files please see our guidelines:

https://journals.plos.org/plospathogens/s/figures

4) We have noticed that you have uploaded Supporting Information files, but you have not included a list of legends. Please add a full list of legends for your Supporting Information files after the references list.

5) Please note that your Data Availability Statement is currently missing the DOI/accession number of each dataset OR a direct link to access each dataset. If your manuscript is accepted for publication, you will be asked to provide these details on a very short timeline. We therefore suggest that you provide this information now, though we will not hold up the peer review process if you are unable.

6) Please amend your detailed Financial Disclosure statement. This is published with the article. It must therefore be completed in full sentences and contain the exact wording you wish to be published.

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

2) If any authors received a salary from any of your funders, please state which authors and which funders..

If you did not receive any funding for this study, please simply state: u201cThe authors received no specific funding for this work.u201d

7) Your current Financial Disclosure states, "Yes ↳ Please add funding details. National Science Foundation (NSF):Ananya Benegal,Michael Vahey 2238165; HHS | National Institutes of Health (NIH):Yuanyuan D He,Zijian Guo,Michael Vahey AI171445 ↳ Please select the country of your main research funder (please select carefully as in some cases this is used in fee calculation). UNITED STATES - US".

However, your funding information on the submission form indicates different funders.

Please indicate by return email the full and correct funding information for your study and confirm the order in which funding contributions should appear. Please be sure to indicate whether the funders played any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

8) Please send a completed 'Competing Interests' statement, including any COIs declared by your co-authors. If you have no competing interests to declare, please state "The authors have declared that no competing interests exist". Otherwise please declare all competing interests beginning with the statement "I have read the journal's policy and the authors of this manuscript have the following competing interests"

Reviewers' Comments:

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: I previously reviewed this manuscript on Review Commons. The authors have done an excellent job of addressing the concerns raised in the previous round of review. The manuscript is much improved, and I recommend it for publication. I have no further comments besides a very minor, optional point for the modeling data in Fig. S3 (please see below).

Reviewer #2: The authors of the PLoS Pathogens manuscript "Antigen flexibility supports the avidity of hemagglutinin-specific antibodies at low antigen densities" present their recent work evaluating antibody interactions with influenza virus proteins. Specifically, they show that the density of the hemagglutinin protein on the surface of the virus impacts antibody binding. Their results show that bivalent antibody binding persists down to one-tenth the density seen on virus particles and that reduced hemagglutinin expression can actually increase antibody occupancy. When hemagglutinin was locked at a fixed angle, the authors were able to show that hemagglutinin flexibility impacts antibody avidity, thereby concluding that the binding of neutralizing antibodies is impacted by surface antigen expression. The authors have responded to comments provided through Review Commons, and their edits and added controls have been incorporated into the manuscript that is under consideration. Below are my thoughts regarding the manuscript and the response to previous comment.

Reviewer #3: In “Antigen flexibility supports the avidity of hemagglutinin-specific antibodies at low antigen

densities”, Benegal et al. develop a microscopy-based assay to measure dissociation of HA head-

binding antibodies from intact virions. This assay allows the authors to explore the contribution of

IgG bivalent avidity to antibody interaction with native virions, which is not accessible using other

methods such as BLI. Using this assay, the authors further explore the effect of HA density on IgG

avidity with engineered low-HA virions and then with artificial HA-coated microspheres. In addition

to measuring antibody dissociation, the authors perform structural analyses to predict the

conformational preferences of many HA IgGs from published structures. The authors conclude that

low HA densities (down to ~10%) still support high avidity binding for the 2 IgGs tested, and thus

there would be little evolutionary pressure for IAV to reduce the HA density as a strategy to evade

immune recognition.

**********

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.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: Authors have clarified comments related to virus selection and morphology, presentation of findings, clarification of key points, and issues related to photobleaching.

Reviewer #3: All my previous comments and those of other reviewers were adequately addressed.

**********

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: Regarding Figure S3, I noticed that cluster 'i' appears to contain nearly all of the HA stem antibodies, plus a few from the head region. Would it be possibe to highlight which data points within this cluster correspond specifically to the stem antibodies?

Furthermore, while this modeling is in the supplemental material, the dataset itself is quite valuable. It would be a great benefit to the field if the authors could make this data available to readers, perhaps in a new supplementary table listing each antibody with its most frequent inter-HA spacing and angle and/or in the authors' GitHub link.

Of note, it also looks like the authors' Github link is still not working, which they may want to fix for the final version of the manuscript.

Reviewer #2: None noted, the data are clearly presented.

Reviewer #3: All my previous comments and those of other reviewers were adequately addressed.

**********

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 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 For information about this choice, including consent withdrawal, please see our Privacy Policy ..

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[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.]

Figure resubmission:

While revising your submission, we strongly recommend that you use PLOS’s NAAS tool (https://ngplosjournals.pagemajik.ai/artanalysis) to test your figure files. NAAS can convert your figure files to the TIFF file type and meet basic requirements (such as print size, resolution), or provide you with a report on issues that do not meet our requirements and that NAAS cannot fix.

After uploading your figures to PLOS’s NAAS tool - https://ngplosjournals.pagemajik.ai/artanalysis, NAAS will process the files provided and display the results in the "Uploaded Files" section of the page as the processing is complete. If the uploaded figures meet our requirements (or NAAS is able to fix the files to meet our requirements), the figure will be marked as "fixed" above. If NAAS is unable to fix the files, a red "failed" label will appear above. When NAAS has confirmed that the figure files meet our requirements, please download the file via the download option, and include these NAAS processed figure files when submitting your revised manuscript.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit 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 Assistant Professor Vahey,

We are pleased to inform you that your manuscript 'Antigen flexibility supports the avidity of hemagglutinin-specific antibodies at low antigen densities' 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,

Alan G. Goodman

Academic Editor

PLOS Pathogens

Thomas Hoenen

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Thank you for revising your manuscript based on the reviewers' remaining minor concerns. I am pleased to recommend acceptance of your manuscript and proceed with the publication process at Plos Pathogens.

Reviewer Comments (if any, and for reference):