Dear Dr. Li,

Thank you very much for submitting your manuscript "The assembly of mutations in 6 genes of HSV1 leads to an attenuated phenotype and induces immunity with a protective effect against viral infection in mice and rhesus macaques" for consideration at PLOS Pathogens. 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,

Pinghui Feng

Associate Editor

PLOS Pathogens

Blossom Damania

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The manuscript # PPATHOGENS-D-19-02372 entitled “The assembly of mutations in 6 genes of HSV1 leads to an attenuated phenotype and induces immunity with a protective effect against viral infection in mice and rhesus macaques” constructed an HSV-1 vaccine candidate M6 with mutations in the UL7, UL41, LAT, Us3, Us11, Us12 genes. The M6 HSV-1 replicates at lower rate and have a small plaque phenotype in culture cells, and an attenuated phenotype in mouse and rhesus macaques. Authors also state that when immunizing mice and monkeys with the mutant strain M6, it induces remarkable serum neutralizing antibody titers and T cells activation, and protect against HSV1 challenge by impeding viral replication, dissemination and pathogenesis.

The significant weekness of this study is that while M6 induces neutralizing antibodies, and reduce the viral replication, the level of neutralizing antibody titer is very low, and M6 does not prevent virus replication in the tissues, and latency establishment in sensory ganglia. The concludion drawn from this study is not in the accordence with the data presented.

Reviewer #2: In this article, the authors established M6 mutants with mutations in the UL7, UL41, LAT, Us3, Us11, Us12 genes as an experimental attenuated vaccine against HSV1. The mutant exhibited attenuated phenotype in an animal model. Furthermore, in mice and rhesus monkeys, the mutant enables to induce remarkable serum neutralizing antibody titers and T cell activation and protect against HSV1 challenge. However, there are some issues to be addressed before further consideration of this ms

Reviewer #3: In this study, the authors constructed an attenuated HSV1 virus strain with mutations in the UL7, UL41, LAT, Us3, Us11, Us12 genes. The animal models infected by the mutant virus, including mice and rhesus monkeys, had a remarkable T cell activation and neutralizing antibody. Moreover, the authors detected the proliferation characteristics and virulence phenotype in different cells and animal models. Overall, the study is important and interesting. However, some more experiments need to be done to strengthen their findings and conclusions.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: No new experiments are suggested, because additional experiment will not make this manuscript better.

Reviewer #2: 1. In addition to detecting neutral antibodies and responsive T cell proliferation in mice or rhesus monkeys, CD4+ and CD8+ T cell responses and of cytokines expression should also be detected by flow cytometry.

2. The authors should take M6 inoculation in mice and rhesus monkeys in a dose-dependent manner to examine indicators of specific immune responses.

3. To better reflect the reliability and authenticity of the research results, the authors should extend the experimental period and continuously observe the changes in neutralizing antibody levels and viral loads.

Reviewer #3: 1.In Figure 1, the authors constructed an HSV1 strain with the assembly of mutations in 6 genes. However, what are the copies of HSV1 genome in every single cell and how the authors make sure that the Us3, Us11 and Us12 genes knockout in all HSV1 genomes.

2.Compared with an HSV-1 mutant vaccine with a UL18 deletion (Acta Virol. 2018;62(2):164-71), what advantages and advances of the M6 mutant vaccine has in cells and animal models.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: The title of this manuscript suggests that M6 mutant induces immunity with a protective effect against viral infection in mice and rhesus macaques; however, the data clearly shows that it does not prevents viral infection in mice or rhesus.

Many sections of figures lack the titles or the details on the y axis.

In the introduction, authors stated that HSV-1 cause occasional genital herpes infection. Epidemiology indicates that more that 50% of first-time genital herpes infections are now caused by HSV-1 virus.

Reviewer #2: None

Reviewer #3: 1.In Figure 1C-G, the authors detected biological properties such as plaques number, growth curves of the M6 strain, M3 strain and WT strain. The authors could also observe the M3 infection of Balb/c mice to further strengthen their conclusions that a more significantly attenuated phenotype for the M6 strain than for the WT strain.

2.In Figure 3, the authors utilized omic analysis of mRNA expression to suggest a characteristic immune response initiated by M6. However, the conclusions seem unconvincingly unless the authors determine the gene expression by qPCR and ELISA.

3.In Figure 4, to confirm their hypothesis that mutation of some virally encoded proteins with functions inhibiting different key molecules in the innate and adaptive immune responses could lead to the host immune system developing an integrated response against viral antigens, the authors should apply flow cytometry technology to detect the T cell activation.

4.In Figure 4 and Table 3, the authors demonstrated that there is no difference between M3 - and M6-immunized mice, suggesting that M3 strain is enough for inducing specific neutralizing antibody and CTL responses. Therefore, compared with M3 strain, please clarify what are the advantages and improvements of M6 strain.

**********

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, PLOS recommends that you deposit 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. For instructions see http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methods

Attachments

Attachment

Submitted filename: Review ppathogens-D-19-02372 2-14-20.docx

Attachment

Submitted filename: Review.doc

Dear Dr. Li,

We are pleased to inform you that your manuscript 'The assembly of mutations in 6 genes of HSV1 leads to an attenuated phenotype and induces immunity with a protective effect against viral infection in mice and rhesus macaques' has been provisionally accepted for publication in PLOS Pathogens. However, I would ask you to modify the title and conclusion according to the first reviewer (see comments below). Please let me know if you need any assistance on this.

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 Pathogens.

Best regards,

Pinghui Feng

Associate Editor

PLOS Pathogens

Blossom Damania

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The revised manuscript # PPATHOGENS-D-19-02372R1 entitled “The assembly of mutations in 6 genes of HSV1 leads to an attenuated phenotype and induces immunity with a protective effect against viral infection in mice and rhesus macaques” designed an HSV-1 vaccine candidate M6 with mutations in the UL7, UL41, LAT, Us3, Us11, Us12 genes. Immunizing mice and monkeys with the mutant strain M6, neutralizing antibody titers and T cells activation, and protect against HSV1 disease.

Reviewer #2: The Authors have addressed all issues raised by this reviewer.

Reviewer #3: (No Response)

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: The data presented in the study show M6 vaccine can prevent mice and rhesus from the HSV-1 disease (Figure 6C). Measurement of virus replication in eye swabs in HSV-1 challenged rhesus, both mock and vaccinated group showed similar level of viral copies (Figure 6F). The data show that vaccine can prevent disease but not the infection. As a matter of fact, the most vaccines against infectious diseases do not prevent infection, however they prevent disease. I propose the title and conclusion of this study should correctly reflect that. Title of this manuscript could be modified to “The assembly of mutations in 6 genes of HSV1 leads to an attenuated phenotype and induces protective immunity and prevent HSV-1 disease in mice and rhesus macaques.” The conclusion should also be revised accordingly.

Reviewer #2: The Authors have addressed all issues raised by this reviewer.

Reviewer #3: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: None

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: None

Reviewer #3: No

Attachments

Attachment

Submitted filename: Review ppathogens-D-19-02372R1 6-4-20 .docx