Dear Dr Buscaglia,

Thank you very much for submitting your manuscript "Deep serological profiling of the Trypanosoma cruzi TSSA antigen reveals different epitopes and modes of recognition by Chagas disease patients" 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. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please pay specific attention to the comments made by Reviewers 2 and 3.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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

[1] A letter containing a detailed list of your responses to all 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).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. 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,

Charles L. Jaffe, Ph.D.

Section Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Section Editor

PLOS Neglected Tropical Diseases

***********************

Please pay specific attention to the comments made by Reviewers 2 and 3.

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

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

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

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

-Were correct statistical analysis used to support conclusions? YES

-Are there concerns about ethical or regulatory requirements being met? NO

Reviewer #2: See Summary and General Comments

Reviewer #3: (No Response)

--------------------

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: -Does the analysis presented match the analysis plan? YES

-Are the results clearly and completely presented? YES

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

Reviewer #2: See Summary and General Comments

Reviewer #3: (No Response)

--------------------

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: -Are the conclusions supported by the data presented? YES

-Are the limitations of analysis clearly described? YES

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

-Is public health relevance addressed? YES

Reviewer #2: See Summary and General Comments

Reviewer #3: (No Response)

--------------------

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: A few minor adjustments are recommended, as follows:

Line 174-175. The authors should clarify in the legend whether the letters ‘A’, ‘C’ , ‘D’ etc, in Figure 1B are or are not the standard single-letter abbreviations for amino acids (as used in Figure 1A).

Line 227. The authors should state (n = 30), rather than simply ‘{30}’.

Line 282. Replace the word ‘above’ with ‘top’.

Line 515-516. Do the authors mean ‘Table 1’ here in the text (not 'Table 2')?

Table 1. In the column headings, the authors should add the superscript numbers to the TSSA peptides as done in the text, for example TSSAIV 24-62

Reviewer #2: See Summary and General Comments

Reviewer #3: (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: This is a detailed analysis of the Trypanosoma cruzi antigen TSSA, revealing new diversity, with relevance to geographical distribution and to diagnostic sensitivity. This rigorous research will contribute to understanding of the epidemiology of Chagas disease, and to improvement of regional diagnostic efficacy.

The methods are complex, and results detailed. However, in the Discussion, some of the results are already described or are expected, for example, as with samples from TSSA1 and TSSAIV, for which it is well-known where these infections predominate. The two variants of TSSA1 are interesting and their epidemiology worthy of follow up, with wider comparative genomics.

There are no significant flaws, and the manuscript is worthy of publication.

Reviewer #2: The authors have a long history in exploring the serological response to the TSSA antigen for the identification of T. cruzi DTU infecting strain.

The manuscript describes a thoughtful work to characterize the linear B-cell epitope profiling of TSSA and reveals variations in the seroprevalence and on the specificity of TSSA antibodies among Chagas disease populations. The findings show a good correlation between the identified TSSA isoform(s) and the reported DTU.

Major points

1. Authors’ citations

- In the manuscript, extensive self-citation of co-authors is noted. Important references are not cited. I invite authors to revise the recent literature. For example, the review by Magalhães et al. 2022 (Lancet Microbe doi: 10.1016/S2666-5247(21)00265-2) on T. cruzi pathogen diversity, immunity, and the fate of infections should clearly be mentioned in the Introduction and Discussion.

- Introduction

Line 103. methods able to reliably assign the infecting strain

Authors should mention that molecular methods are available to genotype the infecting strain DTU and their limitations (see Zingales et al. 2012 MEEGID doi:10.1016/j.meegid.2011.12.009).

- Discussion

Line 530. “as distinct DTUs seem to be associated with differential susceptibility to drugs, disease features and infection outcomes [40]. Reference [40] does not illustrate all the information in the sentence. To this reference must be added [refs 6, 7] and

Zingales 2018. Acta Tropica dx.doi.org/10.1016/j.actatropica.2017.09.017

Magalhães et al., 2022. Lancet Microbe doi: 10.1016/S2666-5247(21)00265-2.

- Reference citation.

The citation of references in the text must be standardized. Sometimes references are cited between square brackets, sometimes between parentheses, sometimes as a superscript.

2. The sera used are a fundamental element of the study. The authors must justify the choice and characterize the patients/sera.

- Justify the six countries whose sera were analyzed.

In the Abstract and in Line 139, it is mentioned “From different endemic settings” and "from different endemic areas". Regions of the USA cannot be considered “endemic”.

Please revise.

- The high number of human sera from the USA (n=18) draws attention.

Most likely the patients are migrants from Latin American countries. This aspect should be mentioned in the article.

- In M&M, Page 6. Line 149, the authors state “The complete information of the population study can be found in [28]”. This reference needs to be updated. Ricci et al. 2023 Nature Communications | (2023) 14:1850.

On the other hand, it is important that the present article reports for EACH COUNTRY the number of the analyzed sera as well as the gender distribution and the mean and standard deviation of age of the patients. It is known that the immune response to T. cruzi varies with these parameters. Please note that for the sera from Argentina the gender of the patients is not specified in Suppl Data 2 by Ricci et al. The sera were provided by Dr. Altcheh, co-author of the present manuscript.

- The authors claim that the patients were asymptomatic (indeterminate ChD form). What tests were performed for this diagnosis? Why is it important for the study that the patients are asymptomatic? Please, inform this in the article.

Reviewer #3: In the present study, Romer et al. performed a deep linear B cell epitope mapping on TSSA (trypomastigote small surface antigen), as adhesin the elicit a strong antibody response during T. cruzi infections. Sera from Chagas disease patients from distinct endemic settings in the Americas, where different parasite DTUs circulate, were used to comprehensively evaluate the reactivity of peptides derived from TSSA sequences from different parasite isolates available in public databases. TSSA isoforms displayed different seroprevalence among Chagas disease patients correlating with differential distribution of parasite DTUs in the Americas. Ala-scan approach allow the identification of critical residues involved in the antibody binding. New TSSA variants and epitopes were identified, including a T. cruzi marinkellei sequence that reacted with serum from a Chagas disease patient from Colombia. This paper represents an important contribution for the study of TSSA linear B-cell epitopes and deserves publication after some aspects are clarified.

Lines 146-149:

Sample naming: What is the letter “E” in the suffix?

Please, provide more information regarding the microarray design, including the complete list of peptides that were evaluated in this study and the raw fluorescence values for each assayed peptide in each experiment.

Provide the public accession numbers for all the TSSA reference sequences (Figure 1).

Legend figure 2: Please state what is the panel “isoforms recognized”

Specify the genome versions as well as the public accession numbers included in the phylogenetic analysis. For genomes not publicly available, please provide the accession number of each gene/protein sequence used in this study or provide all sequences as supplementary material data.

Please provide the nucleotide sequence of the constructs of the (GST)-fusion proteins as supplemental material.

Please include in the Table S2 the database from which each sequence was retrieved.

Please describe the GST-Ag1 (control) used in the Table 1.

Typo:

Line 64: Trypanosoma cruzi: italic

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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

Reviewer #3: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

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.

Dear Dr Buscaglia,

Thank you very much for submitting your manuscript "Deep serological profiling of the Trypanosoma cruzi TSSA antigen reveals different epitopes and modes of recognition by Chagas disease patients" 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. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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

[1] A letter containing a detailed list of your responses to all 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).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. 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,

Charles L. Jaffe, Ph.D.

Section Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Section Editor

PLOS Neglected Tropical Diseases

***********************

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: Revised amendments OK

Reviewer #2: The revised manuscript meets the criteria.

Reviewer #3: (No Response)

--------------------

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: Revised amendments OK

Reviewer #2: The revised manuscript meets the criteria.

Reviewer #3: (No Response)

--------------------

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: Revised amendments OK

Reviewer #2: The revised manuscript meets the criteria.

Reviewer #3: (No Response)

--------------------

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: Revised amendments OK

Reviewer #2: No modifications. Accept revised manuscript.

Reviewer #3: (No Response)

--------------------

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: Revised amendments OK

Reviewer #2: The author responded to points raised by the reviewer and, accordingly, modifications were made to the manuscript.

Reviewer #3: Two points are still pending:

1- The accession numbers of the Figure 1 sequences are still missing (they are not in the legend of Figure 1).

2- Please provide the complete list of TSSA peptide sequences and their raw fluorescence values evaluated in this study as a supplementary file that will be hosted in the PNTD website (and not in the github author’s account).

--------------------

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

Reviewer #3: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

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.

Dear Dr Buscaglia,

We are pleased to inform you that your manuscript 'Deep serological profiling of the Trypanosoma cruzi TSSA antigen reveals different epitopes and modes of recognition by Chagas disease patients' 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,

Charles L. Jaffe, Ph.D.

Section Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Section Editor

PLOS Neglected Tropical Diseases

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