Dear Prof. Pinschewer,

Thank you very much for submitting your manuscript "Adaptive immune defense prevents Bartonella persistence upon trans-placental transmission" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

While two of the reviewers request only minor clarifications, reviewer 3 has requested further dissection of the immune cells/antibody types contributing to the phenotype. While the experiments in points 1, 2, and 4 would increase impact of the work, they could be addressed in the discussion as limitations of the current work. Point 3 regarding the role of milk IgA in elimination of blood infection in WT offspring, however, should be clarified and can be done with existing reagents.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Congli Yuan

Guest Editor

PLOS Pathogens

Nina Salama

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This study explores the role of humoral immune tolerance versus anti-bacterial immunity in vertical/maternal transmission and persistence of Bartonella in mice. The findings of this study reveal a key role for humoral immune defense by the mother and offspring in preventing and eliminating vertical transmission of Bartonella taylorii in mice. These are interesting and novel contributions to the knowledge of the immune response to Bartonella infections.

The questions explored here are well studied and documented. They are of interest to the Bartonella and bacteriology research community, and within the scope of PLOS Pathogens.

Reviewer #2: State of the art story excluding vertical transmission of B. taylorii in mice.

Reviewer #3: In this work, Siewert et al have investigated the frequency of vertical transmission of Bartonella as a function of the ability to generate Bartonella-neutralizing antibodies of both mothers and their offspring. B-cells and antibodies are shown to be required to limit bacteremia and placental bacterial burden in the mothers on the one hand, and is additionally required to successfully fend off infection in the offspring. The data refute a previous model whereby persistent Bartonella infection was due to immune tolerance to the infection of the offspring. The data are strong and the experiments well controlled.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: None

Reviewer #2: - none -

Reviewer #3: While concise, the data as currently presented could be improved by the addition of a bit more detail on the following aspects:

1. What is the role of T-cells? The consistent difference in bacterial burden and transmission efficacy between Rag1-/- and uMT mice suggests that T-cells have a role as well. What is the phenotype of T-cell deficient mice in this system?

2. Which antibody class is most important in humoral defense? Please use ko's specifically lacking IgG or IgA to address this question.

3. How important are milk antibodies in preventing bacteremia in infected offspring? Swapping of litters from infected to naive dams, depriving the offspring of potentially protective IgA in milk, would address this question.

4. Finally, how effective are antibodies in WT offspring of heterozygous uMT parents at successfully fending off transmission?

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: L 120 – the word "mating" is more appropriate than "impregnation".

L 153 – "bacterial loads" should be used instead "bacterial titers".

L 246 – should be suggests.

L – 256 – I strongly suggest the authors to add the words "although not significant" after the phrase "and a similar trend". In addition, in line 258 the authors should change to "….and possibly also in µMT-/- mice but not in WT….", as P=0.7209 is not considered significant (although stated by the authors as a trend) – otherwise it might be considered as overestimation of the test results.

L 262-3 – Authors conclude :" Taken together the present findings demonstrate that B cells represent a dual layer of immune protection against vertical transmission of Bartonellae". Authors should be more accurate regarding their findings as their findings are restricted to B. taylorii in mice. I therefore strongly recommend changing the sentence to the following :" Taken together, the present findings demonstrate that B cells represent a dual layer of immune protection against vertical transmission of B. taylorii, and possibly of other Bartonella species, in mice".

L 286 – authors indicate that "blood was collected from the tail vein" – authors should include the time points, frequency of blood collection and how much blood was drawn from each mouse in each time-point. This information is important as it may affect the general health/stress condition of the mice.

L205-6 – the authors wrote :"Not only does the maternal B cell response restrict transmission." This is a fragment that cannot stand by its own. It should be merged with the next sentence in the paragraph.

L 290 – delete the word days.

L 401 – delete the word as.

Reviewer #2: 1. Introduction is too long - please shorten to ~ 66%.

2. line 268-274: please explain: why was a smR mutant used instead of wildtype bacteria? How was this mutant characterized? Are there any other mutations in this particular strain which might artifically influence the experiments?

3. bacterial load was identified by culture from -80°C frozen samples. Although I know that something like this has been published before, I am critical about this procedure. Did the authors compare these data to data obtaind from fresh samples which underwent direct cultivation?

4. Suppl, Fig. 1: Fig. A and B: please give group sizes. Please wrtite out "EAI" as this abbreviation is difficult to find in the text.

Reviewer #3: none

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Dear Prof. Pinschewer,

We are pleased to inform you that your manuscript 'Adaptive immune defense prevents Bartonella persistence upon trans-placental transmission' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Congli Yuan

Guest Editor

PLOS Pathogens

Nina Salama

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):