Dear Mr. Vang,

Thank you very much for submitting your manuscript "Zika virus-like particle vaccine protects AG129 mice and rhesus macaques against Zika virus" 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.  

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[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. 

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[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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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,

Michael R Holbrook, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Rebecca Rico-Hesse

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: The manuscript describes the efficacy of a Zika virus (ZIKV) virus-like particle (VLP) vaccine in AG129 mice and non-human primate. The authors follow the generation of neutralizing antibody using multiple assays and RNAemia following challenge. The methods outlined in the mansucript are acceptable and clearly defined.

Minor comments-

1. Please define the contents of the “formulation buffer” on line 125.

2. Please provide an explanation for using different alum doses for the non-human primate studies, since the dose of alum remained consistent for each VLP dose in the mouse studies.

Reviewer #2: The Methods section is cluttered and includes redundancies, such as the information about the macaque cohort.

The use of multiple methods to measure neutralization is impressive.

Reviewer #3: Yes. But methods section needs to be written more concisely and with more clarity. Furthermore statistical analysis of different sample groups need to be done, not just compared to mock (alum groups)

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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: The results are easy to follow and match the analysis. The figures are clearly presented.

Reviewer #2: Minor modifications requested:

- Does the isolate of PRVABC59 contain the variants known to be associated with attenuation (Duggal et al, https://doi.org/10.1016/j.virol.2019.02.004)? if so, the authors should cite this paper and state the caveats associated with this virus. The data are still valid as this isolate has been used extensively but this information is important for interpretation.

- some figures represent data post-vaccination but are focused on data post challenge. this is fine, but could be clarified by, for instance, showing the number of days post challenge in parentheses. This is clear in the text, but the figures as they stand are a bit confusing.

Reviewer #3: yes. see attached file for detailed comments.

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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: The data presented supports the conclusions of the manuscript.

Reviewer #2: -The authors state the dose used in the macaque studies (~10,000pfu) could be too low or the species not ideal resulting in inconsistent detection of infectious virus. The discussion would benefit from comparison with published studies as various viral inoculum doses and macaque species (e.g. cynomolgus, pigtail) have been used.

Reviewer #3: yes

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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: Minor comments-

1. In all the figures related to the mouse studies, the 0.1ug VLP + alum group is missing the word “VLP”.

Reviewer #2: (No Response)

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: The study is well written and thought out. While other VLP based vaccines against ZIKV have been tested before in mice, this seems to be the first report in non-human primates. The authors missed an opportunity to show that the antibodies generated following vaccination with this VLP scheme are unlikely to promote antibody-dependent enhancement (ADE), which will be important prior to moving into human trials. Overall, the data supports the conclusions of the manuscript.

General Comments and Experimental suggestions-

1. Are the structural proteins on the virus-like particles produced fully mature or are there a mixture of mature and immature particles? Please discuss.

2. The vaccination schemes generated robust neutralizing antibody responses. It would be interesting to know if the majority of the antibodies generated were type specific and/or bound to domain III of the E protein.

3. Since ADE is of concern particularly in areas with DENV co-circulation, it will be important to show the absence or limited generation of the cross-reactive antibodies or lack of cross-neutralization with DENV.

Reviewer #2: This study is thorough and the manuscript is well-written. Despite the decrease in ZIKV incidence the virus still represents a potential public health threat and vaccines may be needed to stem the spread of a new epidemic should it arise. The use of several methods to measure neutralizing antibodies is impressive and not typical for comparable studies. The use of both mice and primates is likewise impressive. The use of a virus now thought to harbor tissue culture adaptations that result in some in vivo attenuation is unfortunate (if true) but not restricted to this study. However, this information should be included for context as the field moves forward with vaccines and therapeutics.

Reviewer #3: see attached document

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

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Attachments

Attachment

Submitted filename: PNTD-D-20-01817 Review.docx

Dear Mr. Vang,

We are pleased to inform you that your manuscript 'Zika virus-like particle vaccine protects AG129 mice and rhesus macaques against Zika virus' 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,

Michael R Holbrook, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Rebecca Rico-Hesse

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: The authors have addressed the concerns of the reviewers.

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

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: The authors have addressed the concerns of the reviewers.

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

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: The authors have addressed the concerns of the reviewers.

Reviewer #2: (No Response)

Reviewer #3: yes

**********

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)

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: (No Response)

Reviewer #2: Thank you to the authors for carefully addressing my comments and concerns. All concerns raised by this reviewer were addressed.

Reviewer #3: (No Response)

**********

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