Dear Dr. Turano:

Thank you for submitting your manuscript " COVID-19: a complex disease with a unique metabolic signature" for review by PLOS Pathogens. Your manuscript has been evaluated at editorial level and external peer reviewers. The reviewers appreciated the attention to important topic but identified some aspects of the manuscript that should be improved. We therefore request you to modify the manuscript according the review comments before we can consider for acceptance. Your revisions should address these specific points raised by each reviewer.

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).

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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,

Hung Nguyen

Guest Editor

PLOS Pathogens

Sonja Best

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

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Dear Dr. Turano:

Thank you for submitting your manuscript " COVID-19: a complex disease with a unique metabolic signature" for review by PLOS Pathogens. Your manuscript has been evaluated at editorial level and external peer reviewers. The reviewers appreciated the attention to important topic but identified some aspects of the manuscript that should be improved/ We therefore request you to modify the manuscript according the review comments before we can consider for acceptance. Your revisions should address these specific points raised by each reviewer.

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: In this manuscript, Ghini et. al. investigate the metabolic signature of SARS-CoV-2 infection, in relation to viral variants, vaccination status, sex differences and comorbidities and try to identify metabolites as prognostic markers to the fatal outcome of the disease. Finally, by utilizing a multi-organ metabolic model they attempt to simulate the impact of COVID-19 on the entire host metabolism. The authors try to shed more light on the effects of the SARS-CoV-2 infection on host metabolism and improve our understanding of the pathology of COVID-19. The fact that the authors collected samples from a big cohort and through a broad time period strengthens the study. On the other hand, by employing NMR analysis instead of MS they limit the detection of metabolites and are not able to address all the changes in host metabolism. Overall, the concept of the study is interesting as it addresses understudied aspects of the SARS-CoV-2 infection such as viral variants and vaccination effect. However, there are there are some issues on this study that need to be addressed, in order to improve the quality of the manuscript.

Reviewer #2: The manuscript "COVID-19: a complex disease with a unique metabolic signature" by Ghini et al provide novel dataset describing metabolite and lipoprotein signature in realtion to COVID-19 infection. This is a follow up manuscript to previous work published by Authors. The number of samples used for analysis are good and Authors have provided different comparisons like Sex differences, vaccination, etc. Overall this manuscript provides novel information to the field.

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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: 1) In the manuscript it is stated that the samples were obtained at the peak of infection, but it is not clear whether the patients were hospitalized before or after the sampling. Hospitalization and maybe nutrient supplementation, especially for the severe patients and those with fatal outcome, could affect the metabolic signature and in particular the glucose levels. The authors should clarify that the patients received no nutrient supplementation prior to obtaining samples.

2) In figure 8, the authors compare the metabolic activity of fatal patients to healthy controls. Wouldn’t a comparison between fatal and severe (recovered) patients would also give us more indications on which metabolic pathways or organs are more perturbed and could lead to death because of SARS-CoV-2 infection?

Reviewer #2: This work is novel but is very similar to what authors have done previously with ~350 samples. The analysis done are different but the techniques used is similar. So, in future authors should make an effort to make this work comprehensive and with more samples and long duration follow up of the patients if possible.

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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: 1) In the text for figures 1 and 2 the authors state that there are 11/25 metabolites and 16/30 lipoproteins significantly different compared to the control group. But in the figures, the highlighted number of significantly altered metabolites and lipoproteins is different (for example for variant o there are 16 metabolites and 18 lipoproteins highlighted). Why is that? Are the authors only referring to the metabolites changed in all variant groups? That should be made clear.

2) In figure 1, Ornithine is only changed in the o variant group. In the study by Li et. al. (Frontiers in Immunology, 2021) ornithine was found upregulated and correlating positively with inflammation in severe COVID-19 patients. The authors should comment on that, also regarding maybe the less severity of the o variant compared to previous variants of SARS-CoV-2?

3) On the same context, all the ketone bodies are also more increased in the o variant group compared to other groups. The authors should also comment on that.

4) In figure 3, although the colors are explained in the figure legend, it would be helpful to have a color-coding legend in the figure for VAX and NO-VAX groups.

5) In line 282 it is written that “This analysis revealed a clear cut between…” Something is missing.

Reviewer #2: Please confirm and update that samples used were >500 and not >600 as the author summary section says >600.

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Reviewer #1: Yes: Fotios Karagiannis

Reviewer #2: 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.

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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 Prof. Turano,

We are pleased to inform you that your manuscript 'COVID-19: a complex disease with a unique metabolic signature' 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,

Hung Nguyen

Guest Editor

PLOS Pathogens

Sonja Best

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 authors have addressed adequately all the comments raised by the reviewers, therefore improving the quality of the manuscript.

Reviewer #2: (No Response)

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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: (No Response)

Reviewer #2: Thanks to the Author for appropriately addressing the concerns.

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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: (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: Fotios Karagiannis

Reviewer #2: No