Dear Dr. Rodenhuis-Zybert,

Thank you very much for submitting your manuscript "TLR2 axis on peripheral blood mononuclear cells regulates inflammatory responses to circulating, non-infectious immature dengue virus particles" 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. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' 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,

Alain Kohl

Associate Editor

PLOS Pathogens

Ana Fernandez-Sesma

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'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: Dengue (caused by dengue virus DENV) is a major clinical problem without licensed vaccines nor antivirals and so understanding host-virus interactions with regard sensing and inflammation is important and my help design novel safe and effective interventions.

Aguilar-Briseño et al., here describe a follow up study from their 2020 paper linking investigating link between TLR2 DENV sensing and pathogenesis. In their previous study, immature particles were not assessed, which is where is the main novelty of this new work lies. Immature, poorly processed virions are commonly found in vivo and their biological role is poorly understood. Their work across both papers highlights the important but complex role of TLR2 sensing during DENV infection in that acute or chronic stimulation results in distinct outcomes.

Aguilar-Briseño et al use a two-cell model of DENV host interactions looking at DENV challenge of PBMCs or pure cell populations followed by incubation of conditioned media with primary vascular cells, which are heavily involved in DENV pathogenesis. To assess the contribution of immature particles, they produce fully immature virions by infection of furin-defieicnt LoVo cell lines. Additional systems looked at include various HEK reporter lines. Using these approaches, the team show immature virions are sensed by PBMCs and monocytes (and HEK cells) in a TLR2(and coreceptor TLR6/CD14)-dependent manner, which results in production of secretable factors that can activate endothelial cells. Interestingly, prolonged TLR2 activation/DENV particle incubation results in enhanced inflammatory mediator production and vascular activation, possibly linked to TNFa.

This work forms the basis of further studies exploring production of DENV immature virions and kinetics of host immune response during primary and secondary infections. It still remains uncertain whether TLR2 sensing is protective or pathogenic across the spectrum of DENV infection in people.

In general, this is a very well-written and produced study, building on - and extending - solid previous work in a scientifically and clinical relevant arena. This reviewer appreciated the ease of presentation, inclusion of various additional controls and mechanistic focus. The stats appear to be carried out appropriately. The study could have benefited from greater numbers of PBMC donors per experiment, genetic KO models of TLR2 in primary monocytes. However, where appropriate, statistical significance and magnitude of change in primary cells is clear, and combination of neutralizing antibodies and HEK cell reporters allowed mechanistic insight. I have several minor points that would help aid in interpretation of the work.

Reviewer #2: In this well developed manuscript, Aguilar-Briseño and colleagues demonstrate that TLR2 in PBMCs mediates an inflammatory response to immature DENV particles. Using DENV with a significant amount of prM on its surface (prM-DENV), they show that TLR2 signaling is induced in PBMCs exposed to this virus, though not directly infected. Through pharmacological inhibition of distinct steps in the pathway, they further show that NFkB signaling and downstream cytokine leads to endothelial cell responses to infection. Finally, they nicely show that these responses are downregulated through TLR2 shedding. Overall, the manuscript is well written and understandable, the figures are nicely described and the diagrams for experiments and the model are very appreciated, and the experiments are logical.

Reviewer #3: This study is very similar to the author’s prior work which showed that TLR2 on monocytes is activated by circulating dengue virus (PMID 32576819). Previously the authors had not specified whether mature or immature virions can trigger this pathway. Here they show TLR activation of PBMCs by immature DENV and characterize the down-stream signaling that follows TLR2 activation, which is an already well characterized pathway. Like their previous study, they again show that monocytes are the main responders in PBMCs to stimulation by virus (here immature virus). The proportions of mature and immature virus in vivo are not known so it is not clear how consequential this distinction is for physiological function.

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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: One significant concern is the choice of controls across the experiments and the worth of additional variables in interpretation of their results. i) Were the prMDENV preparations purified by ultracentrigfution or are they crude? How would this affect results? Ii) Would additional controls such as media from uninfected LoVo cells or from heat-treated prmDENV from LoVo be helpful? Iii) Furthermore, use of standard mature infectious DENV in some experiments would be useful to compare. Can the authors comment on the merits of these additional controls.

Reviewer #2: The authors primary motivation for the manuscript is the detection of prM-DENV via TLR2, which they demonstrate in Figure 1. They do not compare this response to a response from DENV infection, with less prM or with any other distribution of prM. Because the authors spend significant parts of the introduction and a full paragraph of the discussion describing the viral system, it would benefit the manuscript to have this comparison within the manuscript.

Their data regarding TLR2 shedding shows that samples prior to 48 hpi largely maintain TLR2 (no detectable sTLR2). Do the authors envision that the shedding of TLR2 is gradual? Additional timepoints between 24h and 48h would be informative.

It is surprising that the authors find little interferon in their samples, despite clear prM-DENV detection by the PBMCs. They attribute this to differences in the experimental systems (though their cultures also have DCs) or differences in the virus. Their suggestion that efficient infection leads to detection and IFN production is testable within their system, and would significantly benefit this statement.

Reviewer #3: There is no comparison between mature and immature triggered responses so we cant know if the central claim of the study that immature virus is uniquely triggering this pathway is actually true. This is a control missing throughout the study.

All of the studies presented were obtained using a culture system where supernatants are transferred from DENV-activated PBMCs to endothelial cells. There are no in vivo studies or validation that these responses occur in vivo

Given the fact that it has already been shown that DENV activates TLR2 signaling, most of the results of experiments performed validating cytokine production and NFkB activation etc. are fully expected.

I don’t think the flow cytometry based assay is sensitive enough to establish that there was not virus replication. Negative-strand PCR should be used. Also, this should be done on the HUVEC cells too, not only the PBMCs.

Growing virus using Lovo cells to prevent maturation could be used for some studies, but this is an artificial system and may not represent what actually occurs in DENV infection.

Although it is suggested that TLR2 shedding correlates with results in co-cultured endothelial cells, this was not explicitly shown.

**********

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: Title: remove reference to "circulating" as this refers to in vivo situation, which was not assessed in this study. Indeed, the short title is more accurate in my opinion.

Update author summary - reads very similar to abstract, which is not he object of author summary.

Intro:

Spell out RIGI/MDA5

Add in DENV RNA genome having positive sense polarity

Methods:

Cells section: add that LoVo cells lack furin in the first section listing LoVo (as well as in Viruses section)

Viruses section: is there meant to be a reference for production of prM-DENV in LoVo cells?

PMBCs: state what size well plate was used for your PBMC experiments

Were the same 3 donors used throughout?

When prm denv added, is it ever removed? What is its stability? Have the authors tried adding, removal and washing out the virions?

Are the tlr2 inhibitors in the conditioned media when added the vascular cells? Is it possible this could affect endothelial cell biology?

Often it is not clear the number of independent experiments nor what n refers to (wells or complete experiments on different days). Can the authors update each legend documenting this. Although noted everything seems consistent and no issues found.

The work would have benefited from more primary cell donors as significant variation is noted. On this, as variation is biologically meaningful, is there a possibility we can look at responses in PBMCs across experiments by donor or is it likely to reflect technical differences in experiments? Similarly, is it possible and relevant if these donors have had a previous DENV infection? Was this considered in use of donors?

Results:

Dengue virions : change to DENV virions

Alter: significant (> at least 2 fold) - does significant refer to statistics or to fold change magnitude?

Change the word "tagged" (as in tagged as std) to "referred to herein as".

Change dengue virus to DENV where applicable throughout

Re: role of tlr1, there does seem to be a trend towards lower activation upon its neutralization, although perhaps not statistically or biologically significant

Discussion:

What is sensing prmDENV immature in tlr2 block? Is there an additional sensor, possibly RNA or not, operating here during your setup?

What happens to the immature DENV upon tlr2 sensing? Are the particles internalised and removed?

Reviewer #2: Supplementary Figure 7 (or portions of it) seem rather important to the manuscript and it may benefit the paper to have some of these data in the main body of the text.

The difference in the response at 6h and 48h (Figure 5) is interesting and the authors nicely provide explanations for this difference. Their data would seem to indicate that blockage of TLR2/TLR6/CD14 enhances this late response. Are these data statistically significant?

Reviewer #3: (No Response)

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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: Yes: Connor G G Bamford

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 on PLOS Biology: 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. Rodenhuis-Zybert,

Thank you very much for submitting your manuscript "TLR2 axis on peripheral blood mononuclear cells regulates inflammatory responses to non-infectious immature dengue virus particles" 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. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

While two of the reviewers were positive about the manuscript, the third reviewer still has substantial concerns following revision.

The authors should look at the all comments carefully, and: specifically address the concerns on the comparative analysis in figure 1; clarify the use of Lovo cells and their use; state the numbers of donors as used per experiment and discuss the variability observed; and verify or at least discuss the final comment on validating the lack of infection by qPCR.

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,

Alain Kohl

Associate Editor

PLOS Pathogens

Ana Fernandez-Sesma

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

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

While two of the reviewers were positive about the manuscript, the third reviewer still has substantial concerns following revision.

The authors should look at the all comments carefully, and: specifically address the concerns on the comparative analysis in figure 1; clarify the use of Lovo cells and their use; state the numbers of donors as used per experiment and discuss the variability observed; and verify or at least discuss the final comment on validating the lack of infection by qPCR.

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 authors have addressed all of my concerns and I wish them all the best for publication and their future work.

Reviewer #2: The authors have fully and thoroughly addressed my concerns, as well as the other reviewers', and it is my opinion that the manuscript is sufficient for publication.

Reviewer #3: Whereas the authors described TLR2-mediated activation of monocytes to mature virus in their previous study (PMID 32576819), here they have examined TLR2-mediated activation of monocytes to immature virus. Using pharmacological inhibitors they show that blocking this pathway prevents some of the downstream immune activation. My overall assessment of this revision is that a minor revision was made, when a major revision was needed. Also, the authors did not provide more clarity in the rebuttal document and many questions were answered in a confusing manner when a straightforward answer where to find the improved sections of the text, if any, was needed.

**********

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: N/A

Reviewer #2: (No Response)

Reviewer #3: Major issues remain after revision and some were even introduced with the added data. The addition of infectious virus added in response to Reviewer 3 Question 1 also does not clarify the question about the specificity to prM. Although they have added infectious DENV to Fig. 1 in panels D-E, this is separated from panels B-C and no statistical comparison was made between them that would allow the conclusion that the kinetics are different. Figure 1 legend does not clearly state which serotypes/strains were used and if they are matched for comparison between B-C. No concentrations of virus are provided in the figure legend and it is unclear if the same concentrations of virus were used in B-C versus D-E, so these cannot be directly compared without first establishing the similar inoculation was used and the only difference is prM. (Protein titration and addition of equivalent titers based on that might help). This is a major concern since the author's central conclusion of this manuscript is that immature virus activates inflammatory cascades earlier (on a different time course). I don't think this conclusion has been supported with data yet.

The authors have added LoVo cells to the manuscript methods, but they don’t clearly state if they were used to grow all of the the immature virus in the text. This is very confusing. Since this aspect of the conclusions, that the response is dependent on immature virus is a central conclusion it seems important to do this experiment. If you re-read the response to Reviewer 3 Question 1, this response is not directly answering the question so it's hard to evaluate what the authors have done.

The authors did not address some of the valid reviewer concerns, such as reviewer 1’s request to add more human donors since there was very high variability. That comment was not addressed in the slightest and it was written that 6 donors were used, but this is misleading because there are only 3 in any given experiment.

The authors also don’t validate the lack of infection by negative strand virus PCR, which is a very simple control to do and it’s concerning why they are unwilling to add simple inexpensive controls.

**********

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: N/A

Reviewer #2: (No Response)

Reviewer #3: The new Figure 1C is confusing. The authors should show the time course on the x-axis and use the donors as different colors indicated in the figure legend.

I don't think it's accurate to refer to TLR2 as an "axis".

**********

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: Yes: Connor G G Bamford

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 on PLOS Biology: 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. Rodenhuis-Zybert,

We are pleased to inform you that your manuscript 'TLR2 axis on peripheral blood mononuclear cells regulates inflammatory responses to non-infectious immature dengue virus particles' has been provisionally accepted for publication in PLOS Pathogens.

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,

Alain Kohl

Associate Editor

PLOS Pathogens

Ana Fernandez-Sesma

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):