PONE-D-22-23169Hospitalized and non-hospitalized SARS-CoV-2 vaccinated patients show different frequencies of IFNγ-releasing cells: an exploratory studyPLOS ONE

Dear Dr. Longhini,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Additional Editor Comments:

Reviewers have commented against the acceptance of manuscript in its current form. Manuscript suffers from serious concerns regarding the implemented protocol as well as presentation of the data. Please revise in light of reviewers comments and resubmit accordingly.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I'd like to thank you for asking me to review this interesting paper which explains that 2-doses vaccinated patients requiring hospitalization for COVID-19 show a cellular-mediated immune response lower than non-hospitalized or controls, despite similar antibody titers.

The authors should read these published articles to deep discuss their findings:

-Coppeta L, Ferrari C, Somma G, Mazza A, D'Ancona U, Marcuccilli F, Grelli S, Aurilio MT, Pietroiusti A, Magrini A, Rizza S. Reduced Titers of Circulating Anti-SARS-CoV-2 Antibodies and Risk of COVID-19 Infection in Healthcare Workers during the Nine Months after Immunization with the BNT162b2 mRNA Vaccine. Vaccines (Basel). 2022 Jan 18;10(2):141. doi: 10.3390/vaccines10020141. PMID: 35214600; PMCID: PMC8879462.

-Coppeta L, Somma G, Ferrari C, Mazza A, Rizza S, Trabucco Aurilio M, Perrone S, Magrini A, Pietroiusti A. Persistence of Anti-S Titre among Healthcare Workers Vaccinated with BNT162b2 mRNA COVID-19. Vaccines (Basel). 2021 Aug 25;9(9):947. doi: 10.3390/vaccines9090947. PMID: 34579184; PMCID: PMC8472926.

-Sahin U., Muik A., Vogler I. BNT162b2 Induces SARS-CoV-2-Neutralising Antibodies and T Cells in Humans. Preprint. [(accessed on 11 December 2020)]. Available online: https://www.medrxiv.org/content/10.1101/2020.12.09.20245175v1

Reviewer #2: Thank you for the opportunity to review this interesting manuscript. The authors describe the immune response of hospitalized and ambulatory patients infected with COVID-19 and use healthy individuals as controls. Their main finding was a low level of IFNγ-releasing cells among hospitalized patients and a high predicting value for severe disease. While their findings are of value for future research and detecting patients at risk, there are still some major issues that should be addressed.

Major issues:

#1: Definition of study groups: the authors define the groups as hospitalized and non-hospitalized. However, there are several reasons for admission of COVID-19 patients beside their disease severity. I recommend changing the groups (throughout the paper, title, and conclusion) to severe, mild-moderate and healthy subjects.

#2: Study design: For proper validation of the results and the ability to implement them in future studies, several important issues must be addressed by the authors:

Methods:

- How were patients selected? Was it during their stay in the emergency department?

- At which point in the disease course the blood samples were drawn – was it at admission to the COVID-19 department?

- Did patients with severe disease received dexamethasone (or other steroids) for their disease prior to their inclusion in the study? If so – for how many days?

Results:

- What is the mean time from the vaccines to study inclusion for each group?

- What is the mean time from the PCR and from the first COVID-related symptoms to study inclusion for each group?

- Were any patients with mild/moderate disease deteriorated after being included in the study?

- Any significant comparison between two of the three groups should be addressed in Table 1. Currently the footnote of the table addresses this issue, but it is not mentioned in the table.

#3: discussion: While this section is properly written and presents the main findings, I believe the authors need to further discuss the following ideas:

- What is your theory for the basis of your results?

Do you think the low levels of IFNγ-releasing cells were present in these patients before being infected with COVID-19, which means it can be screened in the wider vaccinated population? On the other hand, do you think these patients had an inappropriate response to the infection (which reflects in low levels of IFNγ-releasing cells, and is also the basis for the study you cite in citation 12) and therefore have severe disease? If so, screening is relevant only among infected patients.

Based on your theory you should relate to previous studies, to the results of the healthy subjects (and their high levels of IFNγ-releasing cells) and to the possible implications in the general population.

- Previous studies describe both high and low levels of IFNγ among patients with severe disease, while this was not discussed by the authors in relation to their findings.

- Levels of IgG-S were previously shown not to correlate with disease severity and outcomes. The authors should address it to support their findings. In this regard, I recommend them to use the following paper which showed similar results for comparison: https://doi.org/10.1371/journal.pone.0268050

- The age of hospitalized patients was substantially older (p value is not everything when it’s a small number of patients). The authors should address this issue as a possible confounder or limitation.

Minor issues:

– In the end of the results, you state “A diagnostic specificity of 100% was observed for s.f.c. > 81.2 x 106…”. If I understand correctly, a lower value than 81.2 is indicative for a severe disease not higher. If so it should be changed accordingly.

Reviewer #3: Manuscript Title: Hospitalized and non-hospitalized SARS-CoV-2 vaccinated patients show different frequencies of IFNγ-releasing cells: an exploratory study

Summary:

This is a short research article that showed that hospitalized (severe) COVID-19 patients (n=15) present significantly lower level of Spike-specific T cells as compared to mild COVID-19 patients (n=15) and uninfected vaccinated controls (n=15) after breakthrough infection. In comparison, there is no significant differences for the IgG antibody titre among the three groups.

While the manuscript is simple, it might add some light on importance of cellular immunity in controlling SARS-CoV-2 infection. However there are large limitations that should be clearly highlighted.

First, this is certainly not the first manuscript that analyze T cell response in patients with severe or mild or asymptomatic SARS-CoV-2 infection. As such the introduction should acknowledge the work on T cell response published by other authors that have already showed presence of T cells not only in Covid-19 but also on asymptomatic individuals ( i.e Sekine, T. et al. Robust T Cell Immunity in Convalescent Individuals with Asymptomatic or Mild COVID-19. Cell 183, 158-168.e14 (2020), Le Bert, N. et al. Highly functional virus-specific cellular immune response in asymptomatic SARS-CoV-2 infection. J Exp Med 218, e20202617 (2021).). Furthermore, the abstract ( and discussion) does does not clarify that the analysis of Spike-specific T cell response was done after infection and not before the infection. As such the work does not provide any data about” correlate of protection from infection” but only analyze the pattern of T cell response after SARS-CoV-2 infection. This should be clearly mentioned.

As such the abstract should be changed . Authors should clarify that the analysis was done after infection. Thus “ In this study we aim to evaluate the spike-specific immune responses in patients requiring or not hospitalization for SARS-CoV-2 infection, after breakthrough infection following two doses of BNT162b2 mRNA vaccine” .

In addition the authors should also acknowledge that their work substantially confirmed in vaccinated individuals previous work that show lower level of SARS-CoV-2 specific T cells in individuals with severe COVID-19 ( I.e. Tan, A., et al. 2021. Early induction of functional SARS-CoV-2-specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients. Cell Reports, 34(6), p.108728. and Chandran, A. et al. Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections. Cell Reports Medicine 3, 100557–100557 (2022).) ).

The authors should also discuss and pointed out that in severe COVID-19, lymphocytes count is decreasing and SARS-CoV-2 T cells can be recruited at the site of inflammation and as such the quantity of circulating SARS-CoV-2 T cells can be reduced. This is why it is important to perform longitudinal early analysis ( as in Tan, A., et al. 2021. Early induction of functional SARS-CoV-2-specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients. Cell Reports, 34(6), p.108728. and Chandran, A. et al. Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections. Cell Reports Medicine 3, 100557–100557 (2022) and not only at single time when inflammatory events might be at their peak. This is why is also important to understand when their T cell analysis was performed .

The authors should also acknowledge that the analysis was focused only on Spike, but since samples were collected from donors who were infected with SARS-CoV-2, instead of looking only at Spike-specific T cell responses, it will be complimentary to also look T cells specific for other SARS-CoV-2 structural (Membrane and Nucleoprotein) and non-structural (ORFs) proteins. This will allow the authors to not only better understand the breath of the T cell responses, which is possibly different among the different cohorts, but also know the better representative magnitude of SARS-CoV-2 T cell response, as cellular immunity is contributed by more than Spike-specific T cells. Multi-antigenic T cell response might play a paramount role in preventing severe COVID-19, so it should be discussed in the context of this study.

Other minor points:

1. The authors should be clearer about the demographics of the patients recruited, particularly the number of days post-infection, number of days post-vaccination and whether the donors have recovered from COVID-19, as they were not clear on whether they are studying acute or convalescent samples.

2. Could the authors clarify on the use of steroid therapy (the statement “since more than 10 days”) or other therapy (i.e. antivirals or antibody), particularly in the “severe/critical COVID-19” cohort.

3. The study could benefit from the availability of clinical parameters describing the patients (degree of peripheral lymphopenia in hospitalized vs non-hospitalized patients, inflammatory markers, etc.)

4. It might be better for the authors to include individual datapoints on top of the box plots for the graphs plotted in Figure 1. As it seems like there are obvious outliers, and that cannot be easily inferred with the current graphs. Plotting individual datapoints will allow the readers to better appreciate the data.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-22-23169R1Severe and mild-moderate SARS-CoV-2 vaccinated patients show different frequencies of IFNγ-releasing cells: an exploratory studyPLOS ONE

Dear Dr. Longhini,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Feb 27 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mohd Adnan, PhD

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor Comments:

Manuscript is significantly improved by the authors. However, there are still some minor concerns raised by the reviewer. Please address these concerns and resubmit accordingly.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: (No Response)

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: (No Response)

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: No

Reviewer #3: (No Response)

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Thank you for the opportunity to re-review this manuscript. The authors have made substantial changes and answered all the issues I've raised. The manuscript has substantially improved. Still there are several issues which should be addressed:

- Many minor spelling and grammar mistake should be revised. For example – in the abstract – few spaces are missing, "discriminating" should be discriminate, and so on. This occurs throughout the text.

- If I understand correctly after the revision – all included patients were diagnosed only upon hospital arrival and not before? If so you should change the sentences describing the first two groups which state "in the previous 10 days". Later you wrote that PCR was positive within 36 hours (results), please state the correct timing in all the places.

Reviewer #3: The authors have addressed my main reservations. The text is more balanced and describes objectively the results obtained.

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Reviewer #2: No

Reviewer #3: Yes: Antonio Bertoletti

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Severe and mild-moderate SARS-CoV-2 vaccinated patients show different frequencies of IFNγ-releasing cells: an exploratory study

PONE-D-22-23169R2

Dear Dr. Longhini,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mohd Adnan, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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