PONE-D-22-33598

Hydroxychloroquine reduces T cells activation recall antigen responses: Implications for COVID-19 prevention

PLOS ONE

Dear Dr. Fowke,

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.

==============================

ACADEMIC EDITOR: 

I agree with comments of both reviewers, however, please ignore the comment #2 of the reviewer #2.

Additionally, please separate the figure legend from the main text. It would be more appreciated if the figure legends are stated in a separate page.

==============================

Please submit your revised manuscript by Mar 31 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,

Janin Nouhin, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"Funding support was provided by the Canadian Institutes of Health Research (CIHR) (OCH # 126275) and S5 386-01 from Grand Challenge Canada (JL). "

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"KRF (OCH # 126275) from Canadian Institutes of Health Research (https://cihr-irsc.gc.ca/e/193.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

JL (S5 386-01) from Grand Challenges Canada (https://www.grandchallenges.ca/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.   

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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

**********

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: Summary :

This study characterizes T cell responses in healthy individuals administered with HCQ over a period of six weeks. The authors demonstrate decreased T cell activation in samples taken at treatment endpoint compared to baseline. In addition, T cells obtained from these individuals after treatment with HCQ seem to be refractory to short or long stimulation with antigen peptides, as evidenced by their impaired cytokine production. All of these observations are in line with previous literature on the impact of HCQ on T cell responses. Altogether, the authors use the results of this study to advocate for better monitoring of SARS-CoV-2 vaccine responses in individuals on long-term HCQ treatment regimens.

General comments :

- There is some literature to suggest that HCQ induces apoptosis of T cells. Therefore, did the authors observe any differences in absolute counts of total T cells, CD4+ or CD8+ T cells or percentages of live/dead cells within the populations gated for T cells, especially at 6 wks post treatment.

- Since CD69 is an early activation marker, did the authors look at its expression on T cells in the 12-hour stimulation conditions? These findings could be included in the manuscipt.

- In the methodology section, the authors describe an HPLC based method to determine concentrations of HCQ in patient samples. The HCQ concentrations at baseline and Wk. 6 could be included in Table 1. Furthermore, did the concentrations of HCQ correlate with CD4 and CD8 T cells at baseline and Wk. 6?

Minor comments :

- Materials and methods could be modified to only include methodologies described in the results, e.g. fresh

cervical mononuclear cells were not described in the analyses.

- Line 250 : Should read “Figure 2C” instead of “Figure 3C”

- Resolution of the figure images should be improved.

Reviewer #2: In this study, the author highlights that hydroxychloroquine (HCQ) treatment used for patients suffering with autoimmune disorders may affect their immune response by reducing the T-cell activation and cytokine production.

Major issues:

1. This study elegantly establishes the relationship between HCQ and immune response, although, no evidence has been provided on HCQ affecting the efficacy of COVID vaccine. Hence, all the text in the manuscript must be corrected to make this point clear.

2. Additional validation studies maybe performed to study the levels of T-cell reduction over time.

3. Result section needs to modified extensively. The author needs to explain the rationale and the interpretation of their observed results.

4. In the discussion section, the author mentions that their findings help understand “how long-term treatment used by people suffering from immune disorders may impact their immune response”. Although, this study has been performed for 6 weeks, so it cannot be called as “long-term” in this manuscript.

Minor issues:

1. Table. 1 needs to be well structured and formatted.

2. Title of the manuscript needs to be reframed appropriately.

Overall, a good attempt at understanding the effect of HCQ treatment on immune response.

**********

6. 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

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Hydroxychloroquine reduces T cells activation recall antigen responses

PONE-D-22-33598R1

Dear Dr. Fowke,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Janin Nouhin, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please address the minor comment of reviewer #2:

Minor comments:

1. If there is any new information added from previous studies in the revised manuscript, the references for that must be added.

Please address the minor comment of reviewer #3:

Minor suggestions:

1. In Sociodemographics section, I suggest adding a sentence when recruitment was conducted. This will help put the study in context with regard to SARS-CoV-2 pandemic.

2. Line 363. I would use a different phrase or word for "uptake."

3. Line 402. I would use a different phrase or word for "in take."

4. Line 429-432. A sentence that is specific would be helpfull in the Conclusions section.

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

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 #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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 #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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 #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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 #1: (No Response)

Reviewer #2: Major comments:

1. The title of the manuscript has been appropriately modified as per suggestion.

2. All the comments for conclusion and result sections have been appropriately addressed.

3. Table. 1 has been accurately revised.

Minor comments:

1. If there is any new information added from previous studies in the revised manuscript, the references for that must be added.

The author has made all the necessary changes based on suggestions from all the reviewers.

Reviewer #3: Kowatsche et al. have presented findings that HCQ affects immune recall to specific antigens in a study conducted over a period of 6 weeks. The resuls are convincing and the summary and conclusions sound. The authots have responded adequatly to previous reviews. I have only minor suggestions.

Minor suggestions:

1. In Sociodemographics section, I suggest adding a sentence when recruitment was conducted. This will help put the study in context with regard to SARS-CoV-2 pandemic.

2. Line 363. I would use a different phrase or word for "uptake."

3. Line 402. I would use a different phrase or word for "in take."

4. Line 429-432. A sentence that is specific would be helpfull in the Conclusions section.

**********

7. 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: Yes: David Joseph Kelvin

**********