PONE-D-22-33512Intranasal challenge with B. pertussis leads to more severe disease manifestations in mice than aerosol challengePLOS ONE

Dear Dr. Barbier,

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1) The authors should make clear the strain used to generate the results linked to the figures and data;2) The control of the use of anesthesia can be of importance when comparing nasal versus aerosol challenges;3) Please, see the comments raised by all the reviewers.

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"1. FHD was supported by the National Institutes of Health grants 1R01AI137155-01A1 and 1R01AI153250-01A1. 

https://www.nih.gov/. The sponsor did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

2. MB was supported by the National Institutes of Health grant 1R01AI14167101A1. https://www.nih.gov/. The sponsor did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

3. K.L.W. received funding from the Cell and Molecular Biology and Biomedical Engineering Training Program funded by the National Institutes of Health NIGMS grant T32 GM133369 awarded to Schaller (PI).  https://www.nih.gov/. The sponsor did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

4. KLW was supported by the NASA West Virginia Space Grant Consortium Graduate Research Fellowship Program, Grant #80NSSC20M0055 (2021-2022). https://www.wvspacegrant.org/. The sponsor did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

5. FHD and MB were supported by The WVU Vaccine Development Center, which in turn was supported by a Research Challenge Grant No. HEPC.dsr.18.6 from the Division of Science and Research, WV Higher Education Policy Commission. https://www.wvhepc.edu/. The sponsor did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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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: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #2: Yes

Reviewer #3: 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: The paper is very important for everyone who works with different types of animal challenges. In the article under analysis, the proposed objective is to evaluate the different types of challenge (intranasal and aerosol). However, I would like to point out some comments and suggestions:

1- In the methodology, it is highlighted that intraperitoneal anesthesia is used intraperitoneally to perform the intranasal challenge, but this procedure does not happen when the aerosol challenge is performed. This anesthesia procedure causes stress to the animals, as well as alters the respiratory flow thus facilitating the administration of the bacterium. In addition, the intraperitoneal anesthesia procedure can develop inflammatory processes during injection, thus stimulating the response of inflammatory cells as well as the production of cytokines and chemokines. Therefore, it would be necessary to have the experimental groups with animals without anesthesia (naïve) and the group that had received anesthesia and evaluated the inflammatory process triggered from intraperitoneal anesthesia in animals challenged with aerosol.

2- In the aerosol challenge methodology, it is necessary to highlight more clearly whether it is performed individually or in groups of animals, and the size of the box that is used, because these factors would be limiting for the concentration of bacteria used, as well as the time that the animal should remain under aerosol.

3- Regarding the presentation of the graphs, it is recommended that the values of the scales be standardized, so that it is possible to compare the results and the groups.

4- Given the proposed objective and knowing the parameters to evaluate the challenge in the sense of even comparing the effectiveness of each type of test, it would be important to evaluate the weight of the animals, as well as the clinical score after the challenge. These parameters are important for evaluating the functionality and health of animal challenge outcomes.

Finally, I would like to congratulate the drawings, especially the experimental design, which made the objective and understanding of the article even clearer.

Reviewer #2: I found the article interesting, with effective experiments to prove the proposed question, with a clear objective, and the conclusion of the article correctly answers the question raised. I recommend publishing the article.

Reviewer #3: It is an interesting work and can increase what we know about the B. pertussis Challenge. I think the statistic were well conducted as it seems that the authors addressed correctly the statistics test.

But I missed one information that for me is crucial to analyses the results and also the conclusion.

The authors describe in the methods two strains of B. Pertussis UT25Sm1 and D420. For me is not clear where they use one or another. If use both in the both assays, is also not clear in the results which result is from UT25Sm1 and D420. In the results and also in the figure 2 which strain were used to generated these results? Please can you clarify and indicates in the methods, in the results and also in the figures which one were used?

As I it is no clear about two strains used, maybe were used one for each kind of experiment, or for each assay, if there are some differences in strain used in each assay, I could not support the conclusion as it can be different regarding the strain used. Even the expression of Bvg+ and hemolytic and colony morphology were the same, one strain can replicate quickly than other, or can lead a high immune response leading to the leukocytosis and pro-inflammatory cytokines differences that cannot be necessarily linked with the rout of administration So, it is crucial for me to identify why you have different strains and where you used each one. This can chance all the way we looked to the conclusions.

So, without this clarification I could not move on in the deep analysis of the conclusions linked with the results. I had marked as minor revision but depend on the clarification of this information it can be major revision. As the authors needs to compare more about two strains like the differences in the virulence factors, also the differences of the reactogenicity of each one that can lead to differences in the response, can be like performing one assay side by side with the two strains and so one...lets clarify first the missing information.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Intranasal challenge with B. pertussis leads to more severe disease manifestations in mice than aerosol challenge

PONE-D-22-33512R1

Dear Dr. Barbier,

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,

Paulo Lee Ho, Ph.D.

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

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 #1: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #3: Yes

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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

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 #1: Yes

Reviewer #3: Yes

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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: All requested comments were answered in an integral and objective manner, showing the requested results and explanations.

Reviewer #3: The authors clarify the missing information’s about the uses of the 2 strains (UT25Sm1 and D420) and put on the missing information in the text as well the legend of the figure which one of the strains they use, and also insert the figure of the experiment with the second strain as well as which one the results are. With this we can see each result and not just believe in it, to extrapolate the results and the increase external validity of the experiment.

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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.

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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 #3: No

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