PONE-D-21-12086

Dietary Gut Microbiota Perturbations Influence Murine Vaccine ResponseDietary Gut Microbiota Perturbations Influence Murine Vaccine Response

PLOS ONE

Dear Dr. Kornerup Hansen,

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.

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PLOS ONE

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Additional Editor Comments:

Hansen et. al present studies which address important aspects relevant to vaccination success. Examination of diet as well as antibiotic therapy are both relevant conditions. There are a few areas of concern which must be appropriately addressed prior to acceptance.

Major concerns:

Figure 2e and Figure 4e though providing the same type of data (IgG response after one vaccination) yet in the different study groups (Study A and C) the vaccinated control and vaccinated gluten Free cohort had statistically different levels of IgG in Study A but not in Study C. The variability of the vaccinated control cohort was also considerably less in Study A. Please address.

Since there was neither a gluten-free nor an antibiotic treatment cohort which were unvaccinated all reference to the gluten free cohort and antibiotic treated cohort must be stated as the "vaccinated" gluten free cohort and the "vaccinated" antibiotic treated cohort for clarity. Sentences such as line 298 "To describe whether the gluten-free diet or the vaccination had an impact on serum..." as well as line 302 "To detect whether antibiotics or vaccination had an impact on immune gene expression" must be restated and analysis adjusted accordingly because there was not a antibiotic cohort that was not vaccinated. For example line 302 could be rephrased to state "To detect whether vaccination in the presence of antibiotics had an impact..."

Verify that the n for each figure is correctly reflected in the number of symbols for each cohort. For example in 4e there appear to be an n=13 in the figure yet in the text the cohort is n=15. Define and explain the number of animals represented in figures 5a and 5b. It appears that the number of animals used in this series of experiments varies and is not explained. Why for example were only 5 animals used in the FoxP3+CD3+CD4 vs the CD25+CD4 panel. In addition lines illustrated where the p value applies must be added.

Provide additional detail on the sample preparation and analysis for flow cytometry either in the methods section or within Table 1; specifically the number of cells stained and the final number of cells within the analysis gate.

For p=0.000 restate as p< 0.005. The images were very pixelated.

The additional findings from Reviewer 1 must also be addressed in your resubmission.

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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: Partly

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

Reviewer #1: Yes

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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: No

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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

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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: In this manuscript Kihl and colleagues have looked at the effect of diet and vaccine response. While this manuscript focusses on the effect of gluten-free diet on many avenues such as gut microbiome, host immune response, tetanus specific IgA and serum cytokines, some questions still remain.

Major concerns:

1. It seems like the mouse experiments used only female BALB/cBomTac mice for their experiments. Please explain why male mice were not used for these experiments?

2. Study A: Impact of gluten on primary and boosting vaccination cohort. While there were two groups fed normal diets (vaccinated and placebo), there is no placebo group for the gluten free diet (Altromin modified). These analyses, therefore do not allow the separation of the effects of gluten-free diet from that of vaccinations alone.

3. It is not clear why samples were collected the same day as vaccination for Study A and C and a few days after vaccination in Study B.

4. Was ampicillin administered throughout the duration of the study? Did these animals get diarrhea? If yes, then did the animals that developed diarrhea show differential gut microbiota and immune response as well as serum cytokines etc.

Minor concerns:

1. The figures are pixelated and very hard to read.

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Reviewer #1: No

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PONE-D-21-12086R1Dietary Gut Microbiota Perturbations Influence Murine Vaccine ResponsePLOS ONE

Dear Dr. Kornerup Hansen,

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.

 Both reviewers note a substantial number of serious concerns that must be addressed before further consideration can be made.

Please submit your revised manuscript by Feb 27 2022 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,

Brenda A Wilson, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

As the second academic editor to review this manuscript, it was difficult to discern where the improvements in the revision were since the second set of reviewers expressed very similar concerns to that of the first round of reviewers as well as additional ones. After reading the manuscript through myself, I would agree with these two reviewers that major revision of the manuscript is needed before further consideration can be made.

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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)

Reviewer #3: (No Response)

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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: Partly

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: No

**********

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

Reviewer #3: Yes

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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: No

Reviewer #3: No

**********

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: The paper describes an observation that mice when fed with a gluten-free diet or treated with ampicillin have slightly lower primary antibody IgG response against a tetanus vaccine, but the secondary Ab response seems normal. The authors conclude that dietary or gut microbiota factors should be considered when testing vaccines. The data in most part are correlative, no mechanistic study is engaged.

(1) Fig5 is problematic, it is not known which two groups are different from each other simply by showing a p-value. The same thing is true for Fig4a,b, and e.

(2) Ampicillin treatment in study B is a little bit too long. Does short-term one-week treatment also have an impact?

(3) Can the author demonstrate that the gluten-free diet-induced microbiota changes drive the lower primary antibody response by fecal transfer type of experiment? Or can the author demonstrate which cell population influenced by the diet reduced the antibody response?

(4) The language can be modified further. p10 L223, please delete the ' mark.

Reviewer #3: General comments

This is the reviewer’s first reading of this article, although it appears it has already gone through one round of reviews. Apologies to the authors if the have already gone through a round of reviews.

The article aims to characterize the impact of microbiome alteration (either through diet or antibiotics) on tetanus vaccine immunogenicity in mice. The article is a nice addition to the literature that suggests that alteration of the intestinal microbiome alters host immune response to both vaccines and pathogens. The authors have performed experiments with a large sample sizes and evaluated microbiome, immune cells, and gene expression resulting in an interesting data set that suggests gluten free-diet and antibiotic administration diminish anti-tetanus antibodies following intial vaccination, possibly through different mechanisms.

Major comments

The reviewer requests a better rationale for why tetanus vaccine was used for this study, given that it is known to elicit consistently high antibody responses across geography and across ages.

Also suggest to reference more of the recent (human and murine) literature on how microbiota alters vaccine immunogenicity.

Study design - Studies A, B and have differing intervals of treatment (antibiotic/diet) and differing timing of prime and boost of vaccination as well as measurement of antibody responses making it difficult to extrapolate results across the studies. The rationale for choices are not clearly described.

Analysis – Given the use of antibody titers, request that the authors repeat antibody statistical analyses using transformed data/geometric mean titers.

Article can often be confusing to read and suggest input from a native English speaker. Some hypotheses in introduction and conclusion suffer from large jumps across subjects without clear delineation of whether data comes from human or animal subjects. Suggest to review and adjust claims accordingly.

Minor comments

Abstract

Line 26 Wording is confusing, isn’t the primary purpose of the study to evaluate the effect of diet ( gluten-free or gluten-containing) and antibiotics (ampicillin or not) on tetanus vaccine immunogenicity? Suggest to adjust wording

Line 28 suggest change ‘lowered’ to ‘was associated with’

Line 29 low-responder not defined in abstract or text, suggest to define if this is a statistical outcome or otherwise remove

Line 29 relative abundance or abundance?

Line 30 remove word massively, change to significantly if this is the case. If hypothesis is that diet altered microbiota which then altered vaccine response, suggest to start with change in microbiota in this sentence

Line 30, suggest to remove ‘similarly’- suggests that antibiotics altered microbiota similarly to diet?

Line 31, add significance for all findings

Line 31, define ‘boosting’

Line 32, what was definition of” influenced’’?

Line 36, suggest to alter sentence, “these results suggest …..”

Introduction –

Line 44 The authors describe diminished vaccine efficacy in low-middle income settings, however tetanus vaccines have never been shown to have diminished efficacy in low or middle income settings. Rather, they have high immunogenicity and good protection globally, with possible higher immunogenicity in low-income settings. Please correct

Line 46 Suggest to use the term low-middle income and high income and not ‘developing”and developed”as terminology

Line 48 – all citations have to do with changing vaccine immunogenicity by genetics and age and do not support differential vaccine performance by geography. Please correct

Line 71, remove the word óbviously’, suggest to make less definitive statements, add “can cause” and add that this study was in mice

Line 74, modify that this is a study that was performed in mice

Line 76, add that this is for influenza vaccination

Line 83, cite literature that shows this

Line 88, citation is not about Bangladeshi children

Study Design

Line 136, what was the rationale for the different timing of tetanus vaccination between the three studies? Both timing of prime following arrival and timing of boost differed between Study A and B as well as the time of measurement of the immunoglobulin response following the second vaccination. For study A, why were antibodies measured 2,5 weeks after vaccine dose 1 but 3,5 weeks after vaccine dose 2

Line 145 and 151, describe when antibody titers were measured in relation to vaccination

Stud C, Line 153, antibody titers are measured 1 week following vaccination, why was this time point chosen? Why was a different interval chosen for duration of diet and antibiotics in relation to stud a and c?

Line 233 – How were titers accounted for in the statistics, why were antibody titers not transformed? Please give rationale or log-transform antibodies/present as geometric mean titers

Lines 258/262, list test used for significance

Line 259 – please define TM7

Line 263 – Phylum should not be italicized

Line 269, which microbiome metric is being given a significance here?

Line 272, what was the impact of antibiotics on weight in study C

Line 276, missing figure legend for figure 1?

Figure 2, e and f, list time of blood draw in relation to vaccination; list timing of fecal sample collection, list timing of vaccination in figure or figure legend. Correct figure label for e/f to be anti-tetanus IgG

Figure 3, list timing of fecal sample collection for microbiome, list timing of IgG in relation to vaccination, list timing of vaccination. Correct figure legends to be anti-tetanus IgG

Line 280/285/294 –is this absolute or relative abundance?

Figure 4e, significance is listed as text but not shown for antibiotics – what has a significance of p= 0.008

Table 2, describe duration of time mice were fed diet (4,5 weeks from Study C or 2 weeks from Study A?) Add n for column control and gluten-free

Line 309, figure 1 does not show a serum sample 2 weeks after the first vaccination for Study B, please adjust.

Line 312, what data/statistical test supports the borderline lower variation. Please list statistical test for all p-values.

Line 316, please indicate which comparison is being made for the significance here

Lines 321-337, please do not report trends, only significant values

Figure 5, please indicate which comparisons are significant in the figure rather than describing them on the figure. Please indicate how many mice were specifically in each group (e.g. n=10/group, not 10 overall?). Please label the individual plots, rather than 5a/b alone. Figure 5B seems to be missing?

Line 367, suggest to specify IgG with anti-tetanus IgG

Line 368, remove reference to ‘low-responder’ unless this term is defined and tested with a statistical bound.

Line 369, suggest to remove dramatic, suggest to specify ‘reduced anti-tetanus IgG following the first tetanus vaccination”

Line 372, inter-individual variation in GM or antibody response?

Lines 372-374, this sentence’s meaning is confusing

Line 383, these findings were associative, do the authors mean, for future mechanistic explanations?

Line 384/393, suggest correlating bacterial abundance to antibody titer/Treg to support such an association.

Line 395, see earlier comment on definition high/low responder

Please provide a limitations section, in which differences in experimental design between the study arms are taken into account. Also please address the limitation of doing this study in mice and looking only at tetanus vaccine. Please address the potential impact of the metabolome as mediator of a microbiome effect as well as other components of the microbiome, such as virome/fungiome that have not currently been measured.

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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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.]

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-21-12086R2Dietary Gut Microbiota Perturbations Influence Murine Vaccine ResponsePLOS ONE

Dear Dr. Kornerup Hansen,

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.

 While some of the previous issues raised by the reviewers were addressed, there remain a number of concerns that still need to be more adequately addressed. In particular, the concerns of reviewer 2 regarding the immune cell responses (figure 5) and gluten-free diets are still valid and should be better addressed.

Please submit your revised manuscript by Apr 14 2022 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,

Brenda A Wilson, Ph.D.

Academic Editor

PLOS ONE

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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)

Reviewer #3: (No Response)

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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: Partly

Reviewer #3: Yes

**********

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

Reviewer #2: No

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: 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: I am not satisfied with the authors response.

The examination of the immune cell responses in Figure 5 needs to be clarified a little bit more.

1) what is the baseline levels of all the immune cell population without vaccination?

2) why the n number is not consistent (e.g., for Treg n=5, while total CD3 T n=10?)

3) how to explain the cell number change differences between spleen and MLN?

4) Pls indicated which two groups were compared when indicated there is a statistcial significance.

In my opinion, a fecal transfer experiment to demonstrate the effect of gluten-free diet on IgG response is dependent on gut microbiota should be performed to strength this manuscript.

Reviewer #3: The authors have addressed the majority of my comments and questions

Small comments:

with reference to the revised track changes text

(page 54) Abstract: line 32, add lower initial vaccine titer

(page 58) line 114: although this vaccine generally has high immunogenicity….

(line 161) ‘we reconsidered this’, suggest to include a rationale here

(lines 176/ 183) body weights of mice … were monitored. What was the impact of antibiotics and diet on weight in studies A/B (this could be confounding)

(line 314/315) describe the direction of the correlation and clarify if correlation was for the phylum or species level.

Line 317 typo, ‘single’

Line 347, Table 2 – list all FDRs in table or indicate which FDR level was used to generate table. Relative repeated twice in table legend

Please review text for typos

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Reviewer #2: Yes: Yugang Wang

Reviewer #3: No

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PONE-D-21-12086R3Dietary Gut Microbiota Perturbations Influence Murine Vaccine ResponsePLOS ONE

Dear Dr. Kornerup Hansen,

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.

 Both reviewers agreed that the revised manuscript was significantly improved. However, Reviewer 2 still has some concerns about the study design and analysis, which really could only be addressed by substantial additional experimentation, which I agree is probably outside the scope of the study since Figure 5 is not a large portion of the overall study. In lieu of that, a title that reflects the manuscript content better and inclusion of additional statistical analysis, as per the reviewer's suggestion, could help address this. But, it will be important to include additional comparative discussion of the results, including caveats and limitations of the study design in light of the results and perhaps suggestions for future studies.

Please submit your revised manuscript by Apr 23 2022 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Brenda A Wilson, Ph.D.

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.

Additional Editor Comments (if provided):

Reviewer 2 still has some issues with the authors' responses regarding Figure 5 and the goals of the study. I think some of this could be addressed by changing the title to be more reflective of the manuscript content (as is, it is a bit sweeping in nature). I suggest something on the order of: "Effect of gluten-free diet and antibiotics on murine gut microbiota and immune response to tetanus vaccination." I think it would also be good for the authors' to include the Dunnett's post hoc test (in addition to what has already been done) and provide a brief comparative discussion of the results, including caveats to the analyses and limitations of the study design.

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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)

Reviewer #3: All comments have been addressed

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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: No

Reviewer #3: (No Response)

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

Reviewer #2: Yes

Reviewer #3: (No Response)

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Reviewer #2: Yes

Reviewer #3: (No Response)

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Reviewer #2: Yes

Reviewer #3: (No Response)

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6. Review Comments to the Author

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Reviewer #2: 1) The authors claim that "Gut Microbiota Perturbations Influence Murine Vaccine Response", but have no intentions to prove their conclusions by providing more experimental data.

2) The authors claim that CD3 T cells are reduced by feeding with a Gluten-free diet in Fig 5A, however, it is really hard to believe that it is a biologically significant reduction at least for the "vacc GF" group, although statistically maybe there is a difference. The two-way ANOVA analysis is mathematically correct, but the way the authors presented it in the current format is hard to interpret.

Reviewer #3: (No Response)

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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.]

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Effect of gluten-free diet and antibiotics on murine gut microbiota and immune response to tetanus vaccination

PONE-D-21-12086R4

Dear Dr. Kornerup Hansen,

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.

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Kind regards,

Brenda A Wilson, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: