Dear Dr. Volmer,

Thank you very much for submitting your manuscript "Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model" 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.

Note that the reviews were mixed with one recommending rejection in large part based on novelty due to the published paper by the TenOever laboratory. The other reviewers and I feel there are some additional interesting (although not completely clear) results that could make this paper a sound addition to the SARS-CoV-2 field. In the revision, you will need to address the following: (a) make sure key experiments have independent biological repeats at separate times; (b) provide greater information and methods on the cytokine assay; (c) perform additional ISH and histological analysis as suggested by the Reviewers and (d) provide more discussion as to the lack of antiviral effect of IFN in some of the experiments and how this relates to prior studies.

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,

Michael Diamond

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: Bessière et al evaluated the potential therapeutic effects of high dose type I interferon treatment against SARS-CoV-2 infection in the Syrian hamster model. One day before or after infection, high dose IFN treatment had a modest effect on weight loss. Starting IFN treatment 3 dpi, no effect was observed. Surprisingly, pre-treatment with type I IFN had no effect on infectious virus titer and viral RNA load. In addition, no significant difference in lung pathology was observed in the pre-treated animals, albeit this may be due to the relatively small number of animals per group. Initiating IFN treatment one day after infection had significant effects on lung pathology 2 dpi, and reduced viral RNA load and infectious virus titer 5 dpi. No effect on weight loss, inflammation or virus titer was observed in hamsters treated with IFN starting 3 dpi. Combined these data are rather surprising and it poses the question, why does type I IFN not protect hamsters from SARS-CoV-2 challenge. The authors demonstrate increased Mx2 gene expression following treatment, suggesting that the universal type I IFN is able to induce an antiviral immune response. Another possibility is that the antiviral immune response is not effective against SARS-CoV-2, or the antiviral immune response is not induced in the target cell of SARS-CoV-2. To distinguish between the two possibilities, the authors should perform their Mx1 RNA-ISH staining on interferon treated animals without SARS-CoV-2 challenge. This allows the authors to provide additional insight as to why type I IFN provides minimal protection and compare the antiviral response between treatment and actual infection. Finally, the authors present some intriguing cytokine and chemokine protein data by ELISA and multiplex array on hamster lung homogenates and sera. If these were available to the general scientific community, this would be a major advance for the field. However, minimal details are provided in terms of product number, antibody clones etc. A search for Syrian Hamster cytokines MilliPlex xMAP assay on the company website yields no hits. AssayGenie appears to have several ELISA kits including IL-6, IL-1b etc., but there is no mention of CXCL10 or CCL2. Additional information must be provided to allow others to make use of these reagents and assays.

Major comments:

• All experiments appear to have been done in one large experiment. The authors should consider repeating key findings in a second experiment.

• Details on the ELISA or bead-array kits are missing. What kind of

• RNA-ISH using Mx1 on IFN treated animals will provide important insight into the reason for the lack of a robust effect of type I IFN administration prior to virus challenge.

• Please discuss the surprising lack of IFN effect on virus titers and inflammation in this model.

Minor:

• Type in the methods section on the hamsters. It appears they are 78 weeks old….probably 7-8 weeks old.

• Provide some more details in the “main text” such as the source and dose the type I IFN used. This can be found in the materials and methods section, but should be referenced elsewhere.

Reviewer #2: ‘Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model’ by Bessière et al. examines the impact of intranasal delivery of universal IFNa to SARS-CoV-2 infected hamsters in either prophylactic or therapeutic treatment models. The article is well written and contains appropriate citations of the recent literature. While much of the data from this manuscript confirms previously published works, new details concerning the timing of treatment are revealed in this manuscript and are important to the field. The most dramatic differences in treatment result appear to be in lung histopathology; it would greatly improve the manuscript if this data was expanded upon with high magnification images and characterization of immune cell infiltrates. Overall, this paper demonstrates modest efficacy of type I IFN treatment in preventing the most serious signs of disease including reduced viral titer in the lung, improved lung histopathology and reduction in some cytokine/chemokine levels. However given the compressed treatment window in small animal models, these results are still significant and applicable to COVID-19 patient treatment.

Reviewer #3: Bessiere et al. use the hamster model of SARS-CoV-2 infection to test the efficacy of IFNa treatment on infection on outcome regarding lung viral loads and pathology. For us it not clear where the novelty of the results are, in particular as very similar data have been presented earlier in the same model (Hoagland et al. Immunity 2021) yet in much greater detail and trialing many more conditions (dose response, full DEG analysis). Furthermore, the authors fail to provide strong evidence for a a clear benefit from an proposed IFN regimen. Obviously hamsters constitute a very stringent challenge model, however, it had been demonstrated before that efficient therapeutic interventions for SARS-COV-2 are in principle possible in hamsters (e.g. Kaptein et al. PNAS 2020, in that case using RdRP inhibitors) and hence a --if at all-- 1log reduction in viral loads and almost no reduction in any of the measured cytokines may not be considered an incremental gain in knowledge towards promising treatment options.

**********

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: Key findings should be repeated in a second cohort of animals

RNA-ISH in Mx1 in IFN treated animals to demonstrate the location and intensity of the antiviral host response.

Reviewer #2: The most significant and consistent results showing an effect of IFN treatment were in the ‘early’ treatment group. Do the authors think prophylactic treatment would have been more effective if daily dosing was used instead of every 2 day dosing? How long does ISG stimulation last after treatment? Prophylactic treatment results are not strong as the Hoagland et al, Immunity paper.

Figure 2A – Figure 2 shows the strongest protection data in the manuscript and more detail to explain these results is key to demonstrating the utility of IFN treatment. Why are the lung sections shown from D5 post infection when there are not significant differences in histology score? Additional, high magnification, histopathology images should be included.

The authors should discuss why titer changes are observed only in the lower airway when strong ISG induction was observed in nasal turbinates.

Reviewer #3: Dose response for (i) virus inoculum and (ii) interferon for infection outcome.

The quantification of lung areas/volumes affected may need more vigorous means for quantification to tease out differences, if at all.

**********

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: Figure 1B – Is weight loss significantly different between IFN treated and sham treated groups of hamsters?

It would be very helpful if the titer data in figure 1C was labeled as being from oralpharangeal swabs and D/E being from the lung.

Figure 2B – significance notations are confusing and don’t seem to match the text. Please clarify if all treatment conditions are significant or if only IFN-early led treatment to significant reductions in histopathology.

Reviewer #3: The luminex assays for hamster cytokines are not described but may be of interest for he general public.

**********

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 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. Volmer,

Thank you very much for submitting your manuscript "Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model" 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. There are just a few remaining editorial and data presentation comments that need to be addressed.

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,

Michael Diamond

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 reviewers have analyzed the Mx1 gene and protein expression in hamsters after IFN treatment. This is very nice data and suggests that SARS-CoV-2 can infect and replicate in cells despite the presence of an antiviral immune response. What they have not done is repeat some of the key findings as requested. While I agree with the reviewers that some P-values are unlikely to change, such as the cytokine induction over mock, others certainly can. For example, the virus titer differences (Fig 1) on day 5 are barely significant.

Reviewer #2: ‘Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model’ by Bessière et al. examines the impact of intranasal delivery of universal IFNa to SARS-CoV-2 infected hamsters in either prophylactic or therapeutic treatment models. The article is well written and contains appropriate citations of the recent literature. While much of the data from this manuscript confirms previously published works, new details concerning the timing of treatment are revealed in this manuscript and are important to the field. The most dramatic differences in treatment result are in reduced weight loss, improved lung histopathology and a reduction in virus titer in the lung. High magnification images and generally improved data presentation have strengthened this manuscript. Overall, this paper demonstrates modest efficacy of type I IFN treatment in preventing the most serious signs of disease including reduced viral titer in the lung, improved lung histopathology and reduction in some cytokine/chemokine levels. However, given the compressed treatment window in small animal models, these results are still significant and applicable to COVID-19 patient treatment. Most importantly, the authors do not overstate their findings and accurately present them as part of our growing understanding of how to treat COVID-19 patients.

Reviewer #3: I may have to excuse for any possible oversight, maybe partially due to a lack of obvious mark-ups in the revised version of the manuscript available to me, however, it is not very clear to what extent and where the authors provide significant new insight in the revised version of the manuscript. The impression prevails that little efforts was made to consolidate previous claims. As a minimal requirement, the wording in title (now claiming a true benefit from IFN treatment) may need to be tempered. E.g. from “is beneficial” to “might confer some benefit” or similar. Here also stating “lack of therapeutic benefit of intranalsal IFN” may be appropriate and as such an important finding.

**********

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 Response)

Reviewer #2: My questions and concerns have been adequately addressed in this revised manuscript.

Reviewer #3: As mentioned before, the importance of the rather small differences regarding any of the parameters measured in infected hamsters (weight evolution, virus loads, RT-qPCR, histology score, cytokine) remains of concern. With this regard, I convene with the Editor and Reviewer #1 that consolidation of data in independent biological repeats may be required, at least for some selected key data; I have the impression that all data on infection outcome were generated from a single batch of animals. I understand the ethical consideration that aims to reduce animal suffering. However, if the authors claim (see line 180-181) that their study has relevance to change clinical practice, independent confirmation should be considered more seriously, including from an ethical point of view either endorsing or not to treat patients that are at high risk of developing severe disease.

**********

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 H&E score in Fig 2 is analyzed with an ANOVA. However, the data appears non-parametric and not normally distributed (day 15, early IFN-a). Please check statistical analysis

Reviewer #2: (No Response)

Reviewer #3: The quantitative scoring for histopathology may be accepted, fully trusting the expert pathologist’s view, however more details on the microscopic parameters and lesions quantified would be appreciated to level up with other studies using the same hamster model. Likewise, regarding my original request for more information on the novel cytokine multiplex assay, I understood the journal’s policy to enforce open science. The multiplex assay used here may be of quite some interest to the field that is in desperate need of new state-of-the-art research tools. More efforts in describing the performance, validation and technical specifications (e.g. list of reagents) of these tools would be very much appreciated.

**********

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, 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. Volmer,

We are pleased to inform you that your manuscript 'Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model' 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,

Michael S. Diamond

Section Editor

PLOS Pathogens

Michael Diamond

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):