Dear Dr. Fungfuang,

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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Kind regards,

Harapan Harapan, MD, PhD

Academic Editor

PLOS ONE

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“Thailand Research Fund, Thailand (Grant No. MRG 6080260).”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in 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?

Reviewer #1: Partly

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: This manuscript explores the serum proteomic characterization of wild stump-tailed macaques with and without dengue virus neutralizing antibodies in Thailand. The study identifies differentially expressed proteins (DEPs) potentially linked to antiviral responses and neurodegenerative processes. The findings may provide preliminary insights into host responses in natural dengue reservoirs. However, there are several major comments that need to be addressed:

Major Comments:

1. Infection with flaviviruses can induce cross-reactive antibodies. The authors need to provide PRNT90 data for other flaviviruses, such as the Zika virus, to confirm that the serum samples are specific to DENV and not influenced by cross-reactivity. The manuscript should clearly state the criteria used to rule out cross-reactivity from other flavivirus infections.

2. The manuscript does not fully explain the rationale for examining serum proteomic profiles in monkeys. Please clarify the benefits of this study and how it contributes to the understanding of the disease. This rationale should be clearly stated in the introduction and reinforced in the conclusion.

3. The differential expression analysis was performed to compare between naive and seropositive monkeys. However, it is a significant limitation that the seropositive group was not further stratified. Could the authors analyze whether there are any differential expressions between monotypic and multitypic seropositive monkeys? The distinct immune responses to monotypic vs multitypic dengue infections might influence host protein profiles.

4. The phrase “sequelae of dengue infection” is used, but the macaques were not clinically characterized. Without longitudinal or clinical follow-up, it is difficult to conclude on sequelae. The wording should be revised to accurately reflect this limitation.

5. The use of 1.2- and 1.5-fold thresholds appears arbitrary. Please justify the selection of these specific thresholds. Please clarify why the 1.5-fold threshold was not used for the initial analysis.

6. The criteria for selecting the nine interesting proteins expressed in seropositive monkeys are unclear. The author states that 582 DEPs were identified, with 560 of these shared between the naive and seropositive groups. Please give a clear explanation of the

“shared between” proteins. Additionally, the rationale for focusing on the nine proteins after filtered methods needs to be explained.

7. The author focuses mainly on upregulated proteins. Downregulated proteins and those uniquely DEPs expressed in naïve macaques also need to be analyzed and discussed.

8. While the discussion provides extensive descriptions of the nine proteins, some interpretations seem speculative. For example, the link between upregulated proteins and neurological complications in macaques is overstated, as no clinical data of macaques were collected. The text should be revised to focus on observed associations.

9. In Figure 6, it is unclear whether the PELs correspond to the results shown in Table 4, especially since naïve samples are shown as 0. The bar graph in Figure 6 does not clearly show whether a significant difference exists between the naive and seropositive samples

Minor Comments:

1. Please add the reference “Additionally, the so-called sylvatic isolate of DENV-1 is not phylogenetically distinct from human-derived lineages, leading to uncertainty about its origins”

2. Please provide specific details about the DENV strains used in the study.

3. Line 262: “Affinity propagation” should be briefly explained.

4. Line 273 The “three filter-based methods” should be briefly stated to clarify the methods applied.

5. The statistical data, such as fold-change values, should be added to Table 4

6. Ensure consistent formatting of gene/protein names (e.g., CDK4, TARS2).

7. Please ensure in how research teams are referenced throughout the manuscript, following conventional citation styles, for example, the format 'Tongthainan, Mongkol (21)' should be revised to a conventional style such as 'Tongthainan D, et al.

8. Line 377 “HS-72” should be briefly explained.

9. Figures should be provided in higher resolution.

10. I cannot access the data deposited from the link https://repository.jpostdb.org/preview/851514434663b785894b48. Please check the access code.

Reviewer #2: Reviewer Comments

Overall

The human serum proteomic profiling in dengue is well-studied, corresponding analyses in non-human primates remain limited. This manuscript investigates the serum proteomic profiles of wild macaques with (seropositive) and without (naive) dengue virus-neutralizing antibodies. However, the grouping of wild macaques with seropositive sera for testing is not presented.

The following points may improve the manuscript:

Introduction

Could you please provide more information on the DENV infections in non-human primates, in term of disease manifestation (Symptomatic/Asymptomatic), ADE, viral transmission?

Methods

Could you please describe more on the locations of baited cages or provide the map, and sex and estimated age of all macaques in the study?

In serum protein preparation section, did you use each serum sample (individual macaque) or the pooled of serum sample (naïve, and seropositive groups)?

Results

In Neutralizing antibody against DENV section, could you please describe more on the sex (confounding factors in proteomic profiles) and estimated age (naïve/monotypic/multipletypic may relate to age) in the study?

In Differential protein identification between two macaque groups, could you please classify more on the naïve/monotypic/multipletypic and the low/moderate/high NT subgroup analysis?

Discussion

Could you please explain why these nine proteins represent potential biomarkers in wild macaques for any aspect (transmission, or severity)?

Reviewer #3: 1. (Introduction): Please strengthen the explanation of the immune response against the dengue virus. Please check and cite the following article: https://doi.org/10.52225/narra.v4i1.309.

2. Please explicitly explain the rationale for choosing a sample size of 32 macaques. Is this sample size sufficient, or is it relatively small? If it is indeed relatively small, you must acknowledge this as a limitation and suggest that these findings should be validated with a larger sample size in the future.

3. Please perform a thorough proofread of the entire manuscript to avoid typos such as "mornitor," "temporaty," and others.

4. Please check the manuscript for the correct formatting of 'p-value'; it should be written in lowercase, not with a capital 'P'.

5. (Materials and Methods, Line 138): Please provide a stronger explanation for why the PRNT90 ≥20 criterion was used as the threshold for seropositivity.

6. Consider adding a table containing the characteristics of the two study groups, such as estimated age, sex, and physical condition. This table is necessary to ensure that the two sample groups are comparable.

7. Please explain explicitly the process used to select only 9 proteins for further analysis from the total of 86 upregulated proteins.

8. The statement that GRIN2C, UCHL1, and MBP proteins indicate neurodegenerative damage is too strong and speculative without direct supporting evidence. This interpretation should be presented as a hypothesis, not a conclusion, as these proteins can originate from various sources (such as peripheral nerves or immune cells).

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Dear Dr. Fungfuang,

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.

Please submit your revised manuscript by Feb 12 2026 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.

• A 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 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,

Harapan Harapan, MD, PhD

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: Overall, The revision is mostly satisfactory, but there are a few minor points that should be addressed.

1. Please check and correct Table 2, as the row for DENV-1/DENV-3 is repeated.

2. Please ensure consistent formatting of gene/protein names throughout the manuscript (e.g., Line 453 refers to Threonyl-tRNA synthetase (TRS), whereas Table 4 uses TARS2; Line 488 uses CDK instead of CDK4)

3. Please ensure in how research teams are referenced throughout the manuscript, following conventional citation styles (Line 451Meuren, Prestes, and Line 455,Jung Park)

Reviewer #2: Serum proteomic characterization of Stump-tailed Macaques (Macaca arctoides) with neutralizing antibodies against Dengue virus in Thailand

Reviewer Comments

This manuscript investigates the serum proteomic profiles of wild macaques with (seropositive) and without (naive) dengue virus-neutralizing antibodies. Unfortunately, the grouping of wild macaques with seropositive sera for testing is not presented.

**********

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.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Serum proteomic characterization of Stump-tailed Macaques (Macaca arctoides) with neutralizing antibodies against Dengue virus in Thailand

PONE-D-25-34021R2

Dear Dr. Fungfuang,

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.

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Kind regards,

Harapan Harapan, MD, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments: