Dear Dr. Warfield,

Thank you very much for submitting your manuscript "Randomized Single Oral Dose Phase 1 Study of Safety, Tolerability, and Pharmacokinetics of Iminosugar UV-4 Hydrochloride (UV-4B) in Healthy Subjects" 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 at least 1 independent reviewer. In light of the feedback (below this email), we would like to invite the resubmission of a revised version that takes into account the reviewer's comments.

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).

Important additional instructions are given below your reviewer comments.

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,

Robert C Reiner, Ph. D

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: As a phase 1a study of a potential antiviral compound, this is well designed and conducted and meets ethical and regulatory requirements.

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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: This is a phase 1a clinical trial of an ascending single oral dose in healthy adults of hydrochloride salt equivalent to 3, 10, 30, 90, 180, 360, 720, or 1000 mg of UV-4 ( N -(9’-methoxynonyl)-1-deoxynojirimycinas (6 subjects per cohort), or placebo (2 subjects per cohort).The study was designed to evaluate the safety, tolerability, and pharmacokinetics of this compound. The dose range tested was not justified by any published evidence of the bio-efficacy of this compound against dengue viruses in any animal model. This fact should be acknowledged and explained in the Introduction and Discussion. The authors should drastically shorten the introduction which should clearly state that this compound has been shown to have antiviral properties in vitro and is expected to exert its value in vivo in humans as an antiviral compound. The authors should understand and convey clearly to readers that in humans, dengue viruses cause pathophysiological responses that do respond to treatment. Dengue virus non-structural protein, NS1, is a direct endothelial toxin responsible for the potentially lethal leakage of fluid from the circulatory system that may lead to dengue shock syndrome and death. The use of the word “treatment” in dengue should be reserved for efforts that restore fluid volume or reverse endothelial damage. An antiviral compound may or may not restore endothelial integrity. Careful clinical evaluations will be required to establish this as a therapeutic outcome. Throughout this manuscript, the pharmacological action of UV-4B should be referred to as “antiviral.”

Specific comments:

243. Remove the word “designed” as it is redundant.

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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: As stated above, this compound should not be referred to as a "treatment" of potentially lethal human dengue virus infections. It is difficult for the reviewer to understand how the authors selected the dosage range for testing with no efficacy data available from an animal model.

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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: Several important suggestions have been made above. Satisfactory responses are crucial to the publication of an acceptable phase 1a study.

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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

Dear Dr. Warfield,

Thank you very much for submitting your manuscript "Randomized Single Oral Dose Phase 1 Study of Safety, Tolerability, and Pharmacokinetics of Iminosugar UV-4 Hydrochloride (UV-4B) in Healthy Subjects" 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,

Robert C Reiner, Ph. D

Deputy 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: Excellent

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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: This is a large phase 1 evaluation of the safety, tolerability and pharmokinetics of a therapeutic immunosugar involving 64 adult subjects who received single oral doses of UV-4 as the hydrochloride salt equivalent to 3, 10, 30, 90, 180, 360, 720, or 1000 mg (6 subjects per cohort), or placebo (2 subjects per cohort). In this well designed and executed study all single doses of UV-4 hydrochloride were well tolerated with no serious adverse events or dose-dependent increases in adverse events observed. References 17 – 20 describing current concepts of dengue epidemiology, pathogenesis, dengue vaccine efficacy and safety could be more authoritative and accessible. (1-5)

1. Sridhar S, Luedtke A, Langevin E, Zhu M, Bonaparte M, Machabert T, et al. Effect of Dengue Serostatus on Dengue Vaccine Safety and Efficacy. N Engl J Med. 2018;379(4):327-40.

2. Glasner DR, Puerta-Guardo H, Beatty PR, Harris E. The Good, the Bad, and the Shocking: The Multiple Roles of Dengue Virus Nonstructural Protein 1 in Protection and Pathogenesis. Annual review of virology. 2018;5(1):227-53.

3. Halstead SB, Russell PK, Brandt WE. NS1, Dengue's Dagger. J Infect Dis. 2020;221:857-60.

4. Halstead SB. Is dengue vaccine protection possible? Clinical Infectious Diseases. 2021;72(https://doi.org/10.1093/cid/ciab282).

5. Halstead SB. Vaccine-Associated Enhanced Viral Disease: Implications for Viral Vaccine Development. BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy. 2021;35(5):505-15.

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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: Excellent

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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: None

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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: Suggested additional useful references for consideration.

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Reviewer #1: 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. Warfield,

We are pleased to inform you that your manuscript 'Randomized Single Oral Dose Phase 1 Study of Safety, Tolerability, and Pharmacokinetics of Iminosugar UV-4 Hydrochloride (UV-4B) in Healthy Subjects' 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,

Robert C Reiner, Ph. D

Deputy Editor

PLOS Neglected Tropical Diseases

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