PONE-D-22-00136Low doses of diarrhoeagenic E. coli induce enhanced monocyte and mDC responses and prevent development of symptoms after homologous rechallengePLOS ONE

Dear Dr. Porbahaie,

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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“L. Ulfman and R.J.J. van Neerven are employed by FrieslandCampina. Other authors declare that there is no conflict of interest.”

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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

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: I Don't Know

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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

Reviewer #2: Yes

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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

Reviewer #2: 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: This manuscript reports a dose-response pilot study investigating which immunological factors are associated with this protection using healthy subjects. I have below comments and questions.

In Power calculation, it’s not clear how n=6 was calculated. How big power was designed to achieve for what outcome or hypothesis test in this study? For this pilot study, power calculation is not necessary. Why do you use Plackett-Burman design? How did you use it? It is not related to this dose-response study. The cited reference 18 has nothing about Plackett-Burman design.

In statistical analysis, it is not clear what stepwise approach was used and why it as needed. What nonlinear function of day and dose were included in GEE analysis? What software was used? Does “Fit of the model” mean “goodness of fit for the model” in your statement? What do you mean “when fit was significant”?

Fig 4C and D may be analyzed using Cuzick’s test for trend.

In Correlation analysis, what statistical method was used for correlation?

In each figure legend, please add what statistical methods were used for analysis.

Reviewer #2: In their manuscript entitled: “Low doses of diarrheagenic E. coli induce enhanced monocyte and mDC responses and prevent development of symptoms after homologous rechallenge, the authors describe a challenge-rechallenge study in human volunteers to assess the protective immunity developed from low vs. high dose challenge of a diarrheagenic E. coli strain. They found that the symptoms and diarrhea induced by the primary challenge to be correlated with the challenge dose, all doses protected the volunteers from symptoms upon rechallenge, with stool frequency decreased in those who received the higher primary dose. Immunological markers of inflammation were greater during the primary challenge, with less inflammation during the secondary challenge, with the exception of dual IL-6 and TNF-alpha secreting PBMCs and monocytes, which increased after the second challenge. The manuscript was well written and easy to read.

I just have a few comments:

Some of the material in the methods section (Figure 1) is better in the results section.

Why were only males included in the study? Why were females excluded?

Minor comment:

Methods, p 13 line 133: if the dietary calcium intake was restricted, was it limited to <500 mg/day? Or is it correct as written?

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Porbahaie Review.docx

PONE-D-22-00136R1Low doses of diarrhoeagenic E. coli induce enhanced monocyte and mDC responses and prevent development of symptoms after homologous rechallengePLOS ONE

Dear Dr. Porbahaie,

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.

Specifically, please address the remaining comments raised the reviewers.

Please submit your revised manuscript by Jan 02 2023 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,

Jianhong Zhou

Staff 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.

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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 #1: (No Response)

Reviewer #2: 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 #1: (No Response)

Reviewer #2: Yes

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

Reviewer #1: (No Response)

Reviewer #2: I Don't Know

**********

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: (No Response)

Reviewer #2: 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 #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Please note, the reference list needs update. The reference 18 need to be replaced to Plackett-Burman’s paper.

Reviewer #2: In their revised paper entitled “Low doses of diarrhoeagenic E. coli induce enhanced monocyte and mDC responses and prevent development of symptoms after homologous rechallenge” the authors have addressed the queries of the reviewer.

This reviewer is comfortable with the changes to the paper. I would remove the sentences explaining why this study does not include women. The reasoning of the authors is not consistent with other challenge studies conducted- there has been no difficulty collecting stool separately from women despite their anatomy, and most women can distinguish abdominal pain from a diarrheagenic infection vs. menstrual cramps.

Minor comment:

Results, Gastrointestinal symptom rating scale:

Line 261: “neither” should be “either”

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: 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.

Low doses of diarrhoeagenic E. coli induce enhanced monocyte and mDC responses and prevent development of symptoms after homologous rechallenge

PONE-D-22-00136R2

Dear Dr. Porbahaie,

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,

Jianhong Zhou

Staff Editor

PLOS ONE

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