PONE-D-20-10246

BCG protects against COVID-19? A word of caution

PLOS ONE

Dear Dr. Kellermayer,

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.

Specifically, please take in account the limitation and advance highlighted by the two reviewers.

We would appreciate receiving your revised manuscript by Jun 18 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled '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.

We look forward to receiving your revised manuscript.

Kind regards,

Pierre Roques, 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 financial disclosure:

'NO - Include this sentence at the end of your statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript'

At this time, please address the following queries:

1. Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.
2. State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”
3. If any authors received a salary from any of your funders, please state which authors and which funders.
4. If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

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

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information

[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: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: No

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: No

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: The article from Richard Kellermayer’s group presents a critical analysis of the recent publication by Miller et al 2020 ref. [15] and proposes an original approach to establish a representation of the daily rates of COVID-19 case fatality (i.e. Death Per Case /Days of the endemic [dpc/d]). However, only strictly identical groups (age/sex proportion, living conditions, COVID diagnosis) could be strictly compared and would better answer the question of the effect of the BCG vaccine on current acute epidemic infection; could you address this question by adding an analysis of equivalent groups (for ex. in urban conditions)? Furthermore, to support the article statements, it is necessary to discuss whether the people who have benefited from BCG vaccination or not are really comparable (age/sex proportion, living conditions, COVID diagnosis), and otherwise to highlight the differences observed. Finally, he possible bias of the chosen representation is not discussed.

In the abstract and elsewhere after, it’s supported that “the use of personal protective equipment (PPE) are the only epidemiologic measures” while the facts exemplified may demonstrate opposite situations: please add also that PPE in our day-to-day lives are efficient when associated with frequent and proper hand washing.

There is too much citations from media and general press (e.g. BBC, YouTube…) in the R. Kellermayer’s article that could be replaced with scientific short communications or opinion views. A lot of references citations of key publications are lacking concerning the current epidemic of COVID-19 both in introduction and in discussion part (for example in the introduction: Lescure FX (https://doi.org/10.1016/S1473-3099(20)30237-1) in parallel to ref. [3] and He X https://www.nature.com/articles/s41591-020-0869-5 in parallel to ref. [4, 5]).

One point that particularly deserves to be developed in this article is the explanation of the interest especially in the BCG vaccination (even extension of use, see thereafter) in the context of COVID-19 epidemic: please explain the scientific rational.

Some evaluations in children of a BCG vaccine cross-protection against several types of microbial or fungal infection were published yet (doi:10.1016/j.cmi.2019.04.020 ; doi:10.3389/fimmu.2019.02806) and these studies are not mentioned or discussed. Likewise, there is no reference to the phase III clinical trials which have recently started (Holland (NCT04328441); Australia (NCT04327206)) to answer the question of the prophylactic effect of BCG vaccine on COVID-19, dedicated to health care, aiming at reducing the symptomatology and considered as an emergency measure and not a lasting solution.

In addition, many applications for prophylactic purposes currently take advantage of the anti-inflammatory response induced by this BCG vaccination, e.g. for type 1 diabetes, bladder cancer, or autoimmune diseases. This rational for use should also be discussed.

Reviewer #2: The authors explored the association between daily rates of COVID-19 case fatality and the presence of universal BCG vaccination.

At the time of submission of their manuscript, there was one preprint reporting a correlation between universal BCG vaccination policy and reduced morbidity and mortality for COVID-19, which has not published yet after peer-review (at least, it is not listed in pubmed as for today, the 2nd of May).

The current analysis defined BCG vaccination data based on the same database as the preprint they refer to. For the death rate data, they used the one reported on April 3rd (instead of March 31st) and they did not exclude countries with less than one million inhabitants.

They analyzed the association between the death rate or the Death Per Case (or case fatality rate)/Days from onset (dpc/d) to account for testing and time bias, and BCG vaccination. They also compared Netherlands, Spain, United Kingdom, France, Belgium, Portugal, and Germany (they called Modern "Colonial Era" countries to colonize America and Africa) to others.

They showed that there is significantly less death/million when BCG was introduced before 1980, but it is not the case when Death Per Case/Days of endemic (dpc/d) is considered. Finally, they reported that dpc/d is significantly higher in Modern Era Colonizer countries compared to others.

MAJOR RECOMMANDATIONS

The choice of 1980 has a cut off for having BCG was defined arbitrary. The authors state that “BCG would have affected the population of a country 40 years old and above [i.e. the population with increased vulnerability towards the SARS-CoV-2 infection]”. In studies reporting a non specific effect of live attenuated vaccines (such as BCG, oral polio, measles or smallpox), like the one published by Christine Benn and Peter Aaby, the heterologous protection against all cause morbidity and mortality is long-lasting, but as compared to the transient immediate defense mediated by the innate effector response. It is not as long-lasting as adaptive immune memory, and in no way it lasts 40 years. Moreover, the non potential beneficial non specific effect of BCG might be modulated with time, and other vaccines for instance. Thus, the authors could focus their analysis on a younger population, rather than the entire population of each of the Top 68 countries in terms of covid-19 induced death, or covid-19 severity.

The year of introduction of BCG vaccination can be one variable to study. However, the authors are certainly aware of the heterogeneity of the vaccine coverage depending on the country. Whether BCG vaccination is mandatory or not can result in different BCG vaccine coverage. In addition, the vaccine schedules and strains of BCG with different immunogenicity can differ between countries for instance. This should ideally be taken into account because it can be confounding, or at least be discussed by the authors.

MINOR RECOMMANDATIONS

Please update the introduction and discussion sections with the very recent literature about:

-The presence of viral RNA found in plasma and urine, and the successfully isolation of infectious viruses from these compartments (as for the presence of infectious particles in feces, there are inherent technical difficulties to isolate virus from feces because of the high risk of contamination of cell cultures with other microrganisms than the virus of interest)

-the transmission through aerosols

-the pathogenesis of SARS-CoV-2 (and not SARS-CoV) in non human primates, including the comparison between n=2 old and n=2 young animals

-there are currently several preprints on observationnal studies and the analysis of the association between BCG vaccination and covid-19, with positive, negative or no correlation

-there are now more than one trial to evaluate whether BCG could reduce the susceptibility to SARS-CoV2 infection and severity of covid-19

Please include a paragraph on the hypothesis as to how BCG could protect, or not, or even worsen the susceptibility to SARS-CoV2 infection and severity of covid-19, with few words on the concept of non specific effect of vaccines and on the new paradigm of trained immunity.

The quality of the figures is too low.

**********

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: Yes: Anne-Sophie Beignon

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-10246R1

BCG epidemiology supports its protection against COVID-19? A word of caution

PLOS ONE

Dear Dr. Kellermayer,

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.

as suggested by the reviewer, it might be of good practice to indicate the last paper about BCG and respiratory infection he highlighted.

Please submit your revised manuscript by Oct 30 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Pierre Roques, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

sorry for the delay but the end of shutdown and return to normal life is sometime quite complicated now;

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

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

**********

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

**********

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

Reviewer #2: I Don't Know

**********

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

**********

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: 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 #2: The authors have addressed the comments from both reviewers.

The goal of their work has been better defined (including by rephrasing the title of the paper and their conclusions). They highlight and alert that global COVID-19 epidemiological data (as well as global BCG epidemiological data?) are not reliable enough to base hypothesis-driven research.

After direct correlation analysis between the year of the establishment of universal BCG vaccination and the mortality rates, expressed as death/million or death per case/days, and using 2 datasets (from the beginning of April and mid of May), they now also used multiple regression analyses. They have included more variables, such as the urban population

percentage, population density, and air passengers, in addition to historic colonization status and median age.

The conclusions from the ongoing clinical trials testing whether BCG could protect against COVID-19 are not yet known, however a recent paper demonstrates that BCG can protect the elderly against respiratory infections (Giamarellos-Bourboulis et al., Cell, 31 Aug 2020). The authors might want to cite this reference as a stronger evidence to test the hypothesis rather than epidemiological data.

**********

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 #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.

BCG epidemiology supports its protection against COVID-19? A word of caution

PONE-D-20-10246R2

Dear Dr. Kellermayer,

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,

Pierre Roques, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thanks to have take in acount all the referee comments. I congratulate you for this nice paper.

Reviewers' comments: