PONE-D-23-35794Antibody reactions of horses against various domains of the EHV-1 receptor-binding protein gD1PLOS ONE

Dear Dr. Lechmann,

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.

Please submit your revised manuscript by Feb 22 2024 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,

Gianmarco Ferrara, PhD, MVD

Academic Editor

PLOS ONE

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. Note from Emily Chenette, Editor in Chief of PLOS ONE, and Iain Hrynaszkiewicz, Director of Open Research Solutions at PLOS: Did you know that depositing data in a repository is associated with up to a 25% citation advantage (https://doi.org/10.1371/journal.pone.0230416)? If you’ve not already done so, consider depositing your raw data in a repository to ensure your work is read, appreciated and cited by the largest possible audience. You’ll also earn an Accessible Data icon on your published paper if you deposit your data in any participating repository (https://plos.org/open-science/open-data/#accessible-data).

3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

5. 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. 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 work aims to identify regions of gD1 that give rise to type-specific antibodies after infection with EHV-1 and/or vaccination. Cross reactive antibodies against gD1 and gD4 are known but knowledge about type-specific gD1 antibodies is limited. For that purpose horse sera from Swizerland and from Iceland, which is considered EHV1-free, were used in a diagnostic ELISA and adapted Luciferase immunoprecipitation system (LIPS) assays. Three antigens consisting of N-terminal gD1 protein fragments plus the full length protein were tested. Full length gD1 LIPS assay detected cross-reactive antibodies, however, gD1_83 fragment in LIPS assay seemed to identify gD1 specific antibodies in horse sera.

This work gives new insights into gD1 regions that can possibly give rise to type-specific antibodies which is important to understand the immune reaction after EHV-1 infection and vaccination. The results might help to improve vaccines which to date are not able to block an infection with either EHV-1 or EHV-4.

The data acquisition, assay validation, and statistical analysis seem sound. Weaknesses can be found in the interpretation of the data, placement into context of previous studies, and occasional spelling mistakes. I recommend the publication of this manuscript in PLOS ONE after revision.

Minor points

- line 57: “ EHV-1 in which the original gD (gD1) had been deleted and replaced by EHV-4 gD (gD4)” I am not sure that this statement of a deletion and replacement of gD is correct. Usually essential genes are not easily deleted in viruses. Please check and provide a reference.

- line 60-61: On what data do you build your hypothesis that type-specific epitopes of gD1 could be important for protection against infection?

- line 70-71: Unfortunately the sentence is unclear and with that the aim. There is probably a typo in the beginning of line 71. I understand the sentence in a way that the aim is to identify gD1 fragments that bind type-specific antibodies that arise after infection. If that is so, I wonder why mostly sera from horses was used that were vaccinated as stated in line 464-465 even more so since it is also interesting to see if specific antibodies are formed after vaccination?

- line 128, 131: You should choose one notation for the Cos cells. “Cos cells” or “COS-7 cells”.

- line 151: You probably meant “comprising” instead of “compromising”.

- line 159: I am a bit confused about this sentence. Was the amino acid sequence of the proteins checked or the nucleic acid sequence of the transfected DNA and how?

- line 254: There is an empty box next to the black filled square which probably should not be there.

- line 280-283: Can you please comment on what influence glycosylation of the protein fragments could have on your assay. Would this influence the antibody binding? And if so, would this have any effect on your LIPS assay?

- line 284-285: Since no gD1 specific antibodies were used in the western blot it might be helpful to verify the fragments by mass spectrometry.

- line 291-293: The figure legend should include the notion of glycosylated gD.

- line 411-412: You state that the gD1 fragments are thought to be secreted into the cell culture supernatant. Can you provide data for this claim? If I understood it right you used only cell lysate for western blotting where all protein fragments were apparently detected. It should be more clear if the presented data was generated with protein from cell lysate or supernatant.

- line 414-417: It is not clear to me why it is a consequence from protein being secreted into cell culture supernatant or not in an artificial system that only full length gD1 will be recognized by cross-reactive antibodies. Can you please comment on that and provide references?

- line 417-420: Is the prediction stated here based on any other study or data? Why would the shortest fragment be the one which is most likely to carry the type-specific epitope? Is this based on structural data?

- line 423-425: Do you have any idea why the gD1_160 did not work so well in your LIPS assay and why the positive sera values varied so much?

- line 442: How do you explain the one “true positive” result from an icelandic horse serum?

- line 458-462: For point 2 and 3 a references are needed. It would be helpful for the reader to have info about the kind of vaccines that are in use and the immune responses that are to be expected.

- line 473: A reference is needed for the statement that type-common antibodies are not protective against EHM.

- line 474: “It is uncertain that...” should be replaced by “It is uncertain if...”.

- Figure 7 and Figure 9 are the same.

- It would be really interesting to try to link the data to the structure of gD1 and gD4 protein in the discussion. It should be taken into account that fragmenting of proteins can result in different molecular structures which can influence antibody binding. A quick modeling via AlphaFold might help to see, if the structure of the gD1 fragments can be expected to correspond to full gD1.

Reviewer #2: Overall:

In this paper the authors investigate antibody responses to the related equine herpesviruses (EHV1 & 4, which exhibit significant sequence similarity). Former can give rise to EHM. What disease/clinical signs does EHV4 elicit?

Serum antibodies were detected via immunoprecipitation assay, to the gD glycoprotein (GP) via a series of N terminal fragments of increasing length. Compared with ‘type-specific’ ELISA. Horse sera from two distant and distinct countries (Switzerland and and Iceland) with different EHV incidence/vaccination.

Generally very well written, scientifically thorough and clear article.

Mostly very good, detailed description of M&M and Results.

Discussion raises some interesting points, some covered in a fulsome manner but other statements are not always followed up as fully as they could be – see recommendations in Minor Comments.

[Clarify if any EHV vaccination is carried out on Iceland – I presume not, as would impact study. I note brief mention in the M&M.]

Other comments which need addressing:

Abstract

Need to state Iceland was chosen as believed to be free of EHV1.

Either Luciferase should be in lower case or immunoprecipitation and system in uppers case (for the LIPS abbreviation) and used throughout.

Introduction

Line 17 – insert ‘respectively’ after gG4 in (gG1 and gG4)

Line 50 – suggest noted that these alternative vaccines used outside Switzerland

Line 68 – the region of non-identical amino acids should also be defined

Line 75 – briefly explain A/G-coated bead system

Line 85 – not that this would be due to cross-reactive antibodies

Materials & Methods

Line 128 – give source and variant # of COS cells and how these were codon optimized (as they are not human cells)

Line 129 – define cmv (also should be upper case - CMV)

Line 148 – ‘scrapped’ should read ‘scraped’.

Line 158/159 – sequencing line should be clarified – maybe ‘coding region’ rather than protein

Line 169 – ‘skimmed milk’

Line 217 – mention Graphpad software company

Results:

Line 245 – It is stated that 21 Swiss and 7 Icelandic sera were gG1 positive (a type-specific ELISA test), however it is then stated that ‘a history of EHV1 was only rarely confirmed’ – this is hardly ‘rare’. Rephrase – lower levels than EHV4 detection?

Line 263-267 are methods rather than results, so should be moved to M&M section.

Line 268 – should read 1-6x

Line 270 – the Fig 3 data for gD1_180 and 402 should be mentioned (for discussion later) as they are interestingly higher than for 160 (noting 160 also gave lower sensitivity result in Fig 5, as mentioned in later Results).

Line 344 – clarify ‘with one exception’.

Line 345 – rephrase ‘clouded’.

Discussion:

Line 403 – whole sentence needs clarifying – from production to transport to secretion (during infection or plasmid transfection or both?). What is cleaved off (and by which protease?). Is the 48aa truly a fragment or a section?

Line 417 – have any antibody epitopes been precisely identified? (e.g. mutation studies?)

Line 423 – authors should discuss why the gD_160 should give such poor responses and yet even 180 gives a reasonable response.

Line 435 – Authors should discuss this discrepancy – were different test assays used? Is monitoring often conducted. Do horses travel internationally from Iceland to events/breeding?

Line 462 – explain why you might see a preference in antibody response against the different antigens in different animals – is this referring to differences between vaccination and natural infection?

Line 472 – are the authors referring to ‘antigenic sin’? If so, how might this impact type-specific vaccination as suggested at the end of the Discussion?

**********

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: Yes: Viviane Kremling

Reviewer #2: 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.]

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.

Antibody reactions of horses against various domains of the EHV-1 receptor-binding protein gD1

PONE-D-23-35794R1

Dear Dr. Lechmann,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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,

Gianmarco Ferrara, PhD, MVD

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 #1: 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

**********

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

Reviewer #1: 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

**********

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

**********

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: (No Response)

**********

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: Yes: Viviane Kremling

**********