Dear Dr. Sangare,

Thank you very much for submitting your manuscript "Individuals co-exposed to sand fly saliva and filarial parasites exhibit altered monocyte function" for consideration at PLOS Neglected Tropical Diseases. 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.

Our sincere apologies for the long delay in this review process, during which ten specialists in the field were successively invited, of which eight unfortunately were unable to review the present manuscript.

Please address the major comments raised by the reviewers, especially the major limitation of the lack of an endemic SG- group which precludes a firm conclusion about the impact of sand fly pre-exposure.

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,

Johan Van Weyenbergh

Associate Editor

PLOS Neglected Tropical Diseases

Brian Weiss

Deputy Editor

PLOS Neglected Tropical Diseases

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: See comments below

Reviewer #2: (No Response)

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: See comments below

Reviewer #2: (No Response)

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: See comments below

Reviewer #2: (No Response)

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Overall, there seem to be very large differences in basal cytokine/chemokine expression of monocytes from the non-exposed (mainly North American, Fig. 1) versus the endemic individuals (SG+/MF+ and SG+/MF-, Fig. 2). Looking specifically at basal IL-6 and CCL4 levels, these fluctuate between 1 and 200 pg/mL in non-exposed individuals versus 100-20,000 pg/mL in individuals from Mali. This is a 100-fold difference in basal cytokine levels, which is neither discussed nor attempted to be explained in the manuscript. The statement in lines 283-284 is misleading as it only refers to SG+/MF+ and SG+/MF-.

On the background of the very high baseline cytokine levels, I do not agree with the conclusion or concept that the cells would have become unresponsive or dysfunctional due to sand fly exposure. The cells seem to be already strongly activated for one or another reason with only a high LPS concentration still having some limited additional stimulatory effect.

Moreover, how can authors attribute differences in monocyte responses to prior sand fly exposure per se in the absence of an SG- endemic control group (which could not be identified in the study area as all individuals are SG+).

As the authors have determined the anti-Pd SG antibody titers as indicators of sand fly exposure, could any correlations be found with monocyte responsiveness? This is probably one of the only options to make a link with sand fly exposure.

Where were the Malian individuals recruited?

How was the blood anticoagulated and cryo-preserved? For improved reading flow, monocyte isolation from whole blood (214-2018) may be moved above the section on in vitro culture. After monocyte isolation, how long were monocytes cultured prior to antigenic stimulation. Was this always the same?

The authors indicate in the method section that antigen exposure was for 30min upto 48h (lines 185-186), but only 24h data are reported and a single experiment with a short 40min exposure for a transcriptional study.

The choice of the recombinant salivary proteins, information on their putative functions and references to the methods of production/purification are lacking in the manuscript. Was the Leishmania antigen always from L. major, this is not clear from the methods section and the various figures. Can the authors discuss the choice and potential limitations of Brugia malayi antigen as filarial antigen in the stimulation assays for a study area where other filarial species occur (Wuchereria, Mansonella), i.e. how comparable are the antigenic repertoires.

In figure 1, it is not clear why so few data points were reported for IL-10 as compared to the other cytokines for the SG Pd stimulated condition. Are data points lacking or are they below detection limit? The legend indicates that each line represents an independent donor (n= 6-8). Based on the number of lines, a quick check seems to indicate that there are up to 14 donors in most groups.

I understand that flow cytometry was done on stimulated/fixed cells of whole blood and on cryopreserved blood. I assume that the same antibody panels were used. Information about the reagents used for intracellular staining seems to be lacking. It is also not entirely clear how the mDCs were identified based on the described panel in lines 223-224. It is clear that additional markers were needed (CD11c+, CD123-, HLA-DR+). For clarity, can authors also indicate the markers used to identify the Tregs and differentiate them from activated T cells. Based on the used panel, I assume that authors can include the B cell frequency as additional information in figure 5 as these also may exert regulatory functions.

Abstract: the first sentence of the conclusions/significance section seems to fit in the above paragraph.

Line 165: what is meant by “eluted” blood

IP-10 and the abbreviation for microfilariae is not consistent (capitalization) throughout text and figures.

Greek letters were not readable in figures 4 and supplemental figure 3.

Reviewer #2: (No Response)

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Attachments

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Submitted filename: Manuscript_final_Review.docx

Dear Dr. Sangare,

We are pleased to inform you that your manuscript 'Individuals co-exposed to sand fly saliva and filarial parasites exhibit altered monocyte function' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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 Neglected Tropical Diseases.

Best regards,

Johan Van Weyenbergh

Associate Editor

PLOS Neglected Tropical Diseases

Brian Weiss

Deputy Editor

PLOS Neglected Tropical Diseases

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