PONE-D-21-18801

The DNA co-vaccination using Sm antigen and IL-10 as prophylactic experimental  therapy, ameliorates nephritis in a model of lupus  induce by pristane

PLOS ONE

Dear Dr. Vázquez-Del Mercado,

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.

Our reviewers found some merits and interests in this study, but also pointed out a number of criticisms that require improvement or amendment. Additional experiments are necessary to answer points raised by reviewers. I ask authors to fully respond to all comments in the revised version. 

Please submit your revised manuscript by Aug 27 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: http://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,

Masataka Kuwana, MD, PhD

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. 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.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

4. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information on the animal research and ensure you have included details on efforts to alleviate suffering.

5. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.  

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

• The name of the colleague or the details of the professional service that edited your manuscript
• A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)
• A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

6. As part of your revision, please complete and submit a copy of the Full ARRIVE 2.0 Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://arriveguidelines.org/sites/arrive/files/Author%20Checklist%20-%20Full.pdf (PDF). Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article.

[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: No

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

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: No

**********

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: No

**********

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: This is an interesting original and novel study that observing the role of Sm-antigen vaccination with IL-10 to induce tolerogenic immunity against lupus. However, several things need to be reworked to obtain a clear conclusion:

1. The vaccination of D2/IL-10 was able to reduce the anti-nRNP/Sm titers, but it was still not known whether this reduction was caused by the D2 or IL-10. The IL-10 alone was already known that could decrease antibody productions due to its immunoregulatory properties. Therefore, the authors should also observe the comparison between the antibody titers after the administration of D2 or IL-10 alone, and the combinations of D2/IL-10.

2. Proteinuria was measured in this study by using a dipstick examination. Rather than using a dipstick examination, which was a semi-qualitative examination, I suggest using quantitative albumin creatinine ratio (ACR) or protein creatinine ratio (PCR) examinations that had better power to explain the extent of proteinuria.

3. The renal pathology was explained in a qualitative approach. Although the authors explained several improvements of the renal pathology findings after the vaccinations, there should be some quantitative measurement that could explain the result more objectively. Several scoring could be used to assess the severity of lupus nephritis, such as NIH Lupus Nephritis Activity and Chronicity Indices.

Reviewer #2: In this manuscript the authors present data suggesting that prophylactic co-vaccination with sm antigen peptides and IL-10 exert a protective role in pristane-induce lupus in Balb/c mice.

There are several issues with the paper that needs to be addressed.

1. It is imperative that all data are shown. For example, figure 2 needs to show data from individual mice as does the bargraphs in figures 4 and 5. Please follow the same standard as used in figure 3.

2. To help the reader, please provide a schematic overview of the animal studies including at what time the mice were injected with what and how many per group. While the information can be pieced together from the materials and methods section it is not easy to read and understand. this is particularly true as only subsets of mice were used for end-of-study analyses (3 mice for kidney analyses, 4 mice for spleen analyses, etc.). Please identify why all studies were not done for all mice, why and how mice for these studies were selected etc.? If the mice came from individual setups (i.e. all mice were not treated with pristane and vaccine at the same time), please indicate each individual cohort of mice and make it clear to the reader when all mice were analyzed together.

3. Please expand the parts of the materials and methods sections that are listed with only a reference. A brief description of the protocol and the specific dilutions used should be written in the manuscript.

4. For proteinuria data, please show individual data as the current bar graphs misleadingly shows that the measure is exact, not based on a dipstick analysis.

5. For kidney analysis, it is unclear if half kidneys were preserved in any solvent/gel such as OCT or the like. As written, the kidneys appear to have been frozen directly. Such protocol would induce water crystallization and tissue damage.

6. Figure 1 shows the presence of specific autoantibodies in sera of co-vaccinated mice. The gel plots do not show recombinant proteins to verify the different specificities. Please include. The gel plots do not show any size ladder to verify protein sizes. Please include. There are 10 samples shown in A and 9 samples shown in B, however, in materials and methods section, it is written that 13 mice were used per group. Please explain or include the remaining data.

7. It is assumed that the percentages given in lines 242-244 and 254-257 are a result of the gel images in figure 1. How is it determined that there is no difference between 54% and 10% as stated? It would be helpful if the authors could present not just the gel plots, but also a graph representing the obtained data.

8. Several sets of data are not supported by the data shown. for example, the authors claim that there are higher levels of IgG2a/IgG1 in mice co-vaccinated with IFNg than in mice co-vaccinated with IL-10 (line 265-267), however, there are no statistical differences between the groups (figure 3). Make sure not to overinterpret the data here and in the discussion.

9. Based on the bargraph appearance of proteinuria data in figure 4 and the highly overlapping error bars, this reviewer is surprised to see statistical significance. Please explain clearly in the figure legend what statistical method was used. As mentioned above, please show individual values rather than the mean/stdev.

10. Figure 5 needs to be redone so that the control samples are represented along with the pristane-treated samples. Please move figure 5A, IFNg-measurements to figure 5B and figure 5A, IL-10 measurements to figure 5C. This will facilitate better understanding of the enhanced production of each cytokine in response to the co-vaccination.

11. The renal histology is very nice and well represented. For comparison, it would be helpful if the authors could solicit a renal score by a renal pathologist blinded to the treatment groups. Also, please provide images from all the mice done - if not in the primary figure, then in a supplemental figure.

12. Similarly, it would be very helpful if the authors could quantify the immunofluorescence stainings either by intensity or by area, underscoring the reduced deposition of immune complexes in IL-10 covaccinated animals.

13. In the discussion the authors write: "We observed that the IgG1, IgG2a levels and IgG2a/IgG1 ratio showed that the prophylactic co-vaccination influences the TH1/TH2 polarization during the course of the disease." This appears to be an overinterpretation of the data as no statistical difference between IgG2a/IgG1 ratios were found. Furthermore, the authors do not provide any evidence for changes in the Th1/Th2 polarization. To support this statement, please provide evidence for altered Th1/Th2 cell levels using either master transcription factors or intracellular cytokines to determine each cell subset.

14. The manuscript needs proof reading for missing words, wrong syntax an typographical errors. If needed, please consult a native English speaker.

-

**********

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: 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.

The DNA co-vaccination using Sm antigen and IL-10 as prophylactic experimental therapy ameliorates nephritis in a model of lupus induced by pristane.

PONE-D-21-18801R1

Dear Dr. Vázquez-Del Mercado,

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,

Masataka Kuwana, MD, PhD

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 #2: (No Response)

**********

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 #2: Yes

**********

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

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 #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 #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 #2: Thank you for responding to my previous comments. The insertion of figure 1 is very helpful. Only a single comment left.

Please make sure that all figure legends adequately describe their figures. this include a basic description of the mice, a notion that each symbol represents one mouse, the n for each group and the statistical method used. This is done for most legends now, but is still missing from a few.

**********

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 #2: No