PONE-D-22-24848Distinct and shared B cell responses of tuberculosis patients and their household contactsPLOS ONE

Dear Dr. Aggarwal,

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 Nov 18 2022 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,

Juraj Ivanyi

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. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

4. 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.

[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: This original research article characterised the B cell responses in tuberculosis (TB) patients and their household contacts, with the view of determining whether these responses can distinguish between latent and active TB disease. The authors recruited smear-positive TB patients and their disease-free household contacts. Serum samples were acquired to determine antibody levels against various Mycobacterium tuberculosis (Mtb) membrane antigens. Whole-blood samples were also taken to characterise populations of B cell subsets by flow cytometry. Briefly, the study found that TB patients had higher levels of IgG against Mtb membrane antigens, whereas household contacts had higher levels of IgM. Interestingly, the authors found differences in the reactivity of TB patient and household contact sera against Mtb membrane antigens which was visualised by immunoblotting. Furthermore, the authors found differences in the frequencies of classical memory B cells (CD19+CD27+) and between TB patients and their household contacts, while frequencies of IgA+ atypical memory B cells (CD19+CD27-) were higher than IgG+ in both TB patients and household contacts. The article concludes that the differences in B cell responses between TB patients and household contacts observed could play a role in a protective immune response against TB, while providing useful information for correlates of protection in vaccine development.

Overall, the article was well-written with a good quality of English and ample detail was provided regarding the methodology. The absence of a “true” negative control group was concerning. While the conclusions made in this article are still valid despite the lack of this control group, a stronger case could be made if this data could be included.

Major comments

• The data and figures in this study would benefit greatly from inclusion of negative controls (samples from TB negative individuals who are not household contacts of active TB patients, whether BCG vaccinated or not). This would allow for further discussion of confounding factors such as exposure to environmental mycobacterial antigens. It would also add weight to specificity argument when discussing immune reactivity to different protein fractions of membrane proteins.

• The authors should discuss the implication of BCG status (I presume most subjects had a BCG scar) on their interpretations of data

• I appreciate the authors’ comments about their data being useful in the context of diseases progression and vaccine induced protective immunity. Is it not, however, that some of the higher molecular weight proteins might be the candidates for serologically distinguishing TB cases and latent TB (diagnostic value)?

Minor comments

• Line 60. Sentence should read “The role of antibodies in protection…”

• Line 176. Include make/model of plate reader used

• Line 212. Include version number for GraphPad Prism

• Table 1. Check spelling, 2 should read “GeneXpert”

Reviewer #2: In this paper, the authors evaluated, in a cohort of tuberculosis patients and their smear-positive household contacts, the humoral adaptive immune compartment by measuring Mtb-membrane-specific antibody levels. Moreover, they also evaluated the B cell subsets in peripheral blood.

Results showed in TB patients a significantly higher level of IgG than IgM and IgA whereas IgM and IgA antibody levels were comparable. In HHC sera had significantly higher IgM antibody levels than IgG and IgA. The ratio of IgM to IgG antibodies in HHCs was also significantly higher than in TB patients. Immunoblotting displays that HHC and TB patients shared the ability to recognize specific low-molecular weight MtM antigens. About B cells, the classic memory B Cells Frequencies were significantly higher, and IgG+ cMBCs were significantly lower in HHCs than in patients.

The authors conclude that IgM/IgG antibody ratio, antibody binding to selected MtM antigens and relative frequencies of MBC subsets could indicate protective or pathogenic immune responses following the primary infection with Mtb.

In my opinion, the paper is well written and gives interesting ideas about the still controversial role of humoral response in Mtb infection and tuberculosis.

I have some points to observe about the results and discussion.

In the legend of figure 1 and 5, the authors should add the statistic test used.

About the statistic, considering the number of patients and HHC enrolled, the authors can show data using the mean and not the median and can use parametric statistic tests if they have a normal data distribution.

The period between lines 298 and 304 is not fully clear. For instance, the authors write that the median frequency of cMBC is 1.8 in HHC and 0.95 in TB patients, but after they write that the frequency of IgG+ cMBC is 1.6 in HHC and 3.0 in TB patients, the last value is higher than 0.95. In my opinion, even if they show a gating strategy, the authors should better specify from which populations the reported frequencies are counted or should report the frequencies to the whole B cells population.

Line 349, are the incidence results based on one hundred thousand subjects per year? Please specify.

Line 353 have the authors checked if there is any correlation between the TST responders among HHCs and the concentration of IgM or IgG or IgA or any other parameter related to B cells?

Line 358, the authors used the LTBI abbreviation for the first time in the paper; they should give the full words of this abbreviation.

Line 365, in literature, there are many discordant data about the concentration and role of antibodies and B cells in tuberculosis or TB infection. Therefore, the authors should give a more thorough background on this point.

Line 407, the authors write: “the HHCs showing an antigen-recognition pattern similar to TB patients could be harbouring an elevated burden of infection.” This is an interesting point. Do the authors plan to verify this hypothesis?

**********

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.

Distinct and shared B cell responses of tuberculosis patients and their household contacts

PONE-D-22-24848R1

Dear Dr. Aggarwal,

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,

Juraj Ivanyi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: