PONE-D-19-27417

Immune responses to Mycobacterium tuberculosis membrane-associated antigens including alpha crystallin can potentially discriminate between latent infection and active tuberculosis disease

PLOS ONE

Dear Dr Sinha,

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: 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

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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

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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: The authors have investigated antibody responses to alpha-crystallin and an Mtb cell membrane fraction in a range of individuals with presumed varying exposure to Mtb, as well as active TB and cured TB.

Major comments

1. Clinical cohort description:

i. How was ‘cured TB’ defined?

ii. Presumably none of the patients with active TB were culture positive, and no drug sensitivity testing was performed?

iii. From the referenced paper describing the cohort, it appears that several of the participants in the active TB group were on antitubercular therapy. Is this the case for the samples interrogated here? Although <3 weeks, this is still sufficient time for sputum conversion to occur.

iv. What is the distribution of TST positivity in the HCW groups? For example, groups are divided into OC and HC, but how many of each of these groups are TST+?

v. Why is the data rather not represented then as TST- HCW, and TST+ HCW, as these groups may be more immunologically distinct?

vi. What is meant by ‘suspected, or had tested for HIV?’ How many were tested, and what were the results? If HIV status is unknown, then this should be simply stated.

vii. If the assumption is that the intensity of exposure is greater in previous household contacts than occupational contacts, is the duration of time since exposure known?

2. Methods:

i. The concentration of secondary antibodies should be given in the methods.

2. Statistical analysis

i. Student’s t-test was used with the presumption of normal data distribution. What is the evidence for selecting a parametric test for this ELISA dataset?

ii. For multiple groups, statistical tests with multiple comparison should be used.

iii. It is not indicated in the figure legends where each statistical method listed is applied. For example, for the correlations, no statistical test is mentioned.

3. Figures:

i. The figures supplied are too low resolution to be clearly read. Please provide higher resolution images of figures.

ii. The figure legends would be improved by stating the statistical methods, numbers and quantitative results obtained. It would also make the figures clearer to interpret if statistical significance was included in the figures.

4. Results:

a) Figure 1:

i. Are there any significant differences using multiple comparisons for each Ig, across the 4 groups and 2 antigens?

ii. How was the inference made that IgG was the predominant isotype? The assays used appear semi-quantitative, a limitation of ELISA without a standard curve. They also utilize different secondary antibodies at different concentrations.

b) Figure 2

i. The conclusion that non-specific serum binding is ‘strikingly’ greater than antigen binding for IgM appears to be a technical challenge rather than biological.

ii. The results that IgM are higher in background wells than antigen-coated wells suggests that IgM is not detected at that concentration of sera, and should be stated as such.

iii. Comparing blocking buffers is reasonable, however this did not resolve the issue. Further optimization of antigen concentration, serum and secondary dilution could improve detection specific IgM.

ii. Prior to describing this as a “striking” finding with “unknown mechanism”, could the authors please review the optimization of this IgM assay, or perhaps include positive controls of known IgM responder serum.

c) Figure 3A and 3B:

i. Please revise figures to include statistical tests, p value and numbers.

ii. How is the lack of correlation to be understood, given that Acr is likely a component of the more immunogenic MtM antigen fraction?

iii. Can the ratio of OD be meaningfully interpreted, given that the OD is a semi-quantitative measure, and MtM is at a higher concentration? It could be understood that these ratios are from technical aspects of the assay rather than biological.

b) Figure 4:

i. The avidity experiment is an interesting approach, given observations in a recent publication (Lu et al, IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure, Nature Medicine, 2019).

ii. What is the specific p value on this figure?

iii. Has a concentration curve of urea, per the above article, been tested? Perhaps this could reveal avidity differences in the contacts vs active TB group.

a. It would be interesting to see if an inverse trend existed for cytosolic antigen fraction.

d) Figure 5 and 6:

i. A representative complete gating strategy should be shown, including a live-dead stain.

ii. The positive population for Ki67 seems to be placed quite high and underestimate stimulation, especially in the active TB panel.

iii. The gate should be placed closer to the negative population with gating derived from a secondary antibody FMO panel shown in the figure. Could the authors please show an FMO figure where this gating was validated? This may even improve the results of the assay.

iv. Could the authors show the raw data analysed as frequency of live Ki67+ T cells.

v. It is unclear what is on the Y-axis. What is meant by ‘responder T cells?’ The figure insert is also illegible.

vi. For the stimulation in this assay, is it possible that more antigen-specific cells are present in ‘cured TB’ and active TB, but these cells die during the 5 day stimulation?

e) Figure 7:

i. It is unclear which samples are these – are they all the OC and HC samples pooled?

ii. The axes do not include antigens tested.

iii. What are the numbers in each group?

iv. What is the cut-off for TST, and how was this determined if HIV infection was not determined?

f) Figure 8:

i. What is the concentration of MtM used in the stimulation assay? Is it fair to conclude that more cells respond to MtM when perhaps non-saturating levels of Acr were used, and the concentrations differed?

Minor comments

1. Single quotation marks appear numerous times in the article, perhaps to make the point that some of these notions are contentious in the literature. These could serve to be removed and the point made simply for readability. Ie. it is not needed to put ‘blocking’ buffer in quotation marks for example.

2. What is meant by a front-line antigens?

3. The authors repeatedly preface their results with several citations and literature review in the results section. This information may be more appropriate in the discussion section.

4. The language includes colloquialisms such as ‘turned out to be’ that seem inappropriate per standard academic convention.

5. Could the authors please revise for grammatical errors.

Reviewer #2: In recent years there are several reports about immunological methods to identify TB infected people and to differentiate active TB from latent TB infection. This paper analyze the antibody response as well as T cell response against M. tuberculosis membrane-associated antigens and the protein Acr.

Introduction is well written and enough information has been included to understand the problem. I consider the methods were designed and performed adequately. Still, I have some general comments that authors should consider to improve the article.

Although he actual immunological methods cannot discriminate between active TB and latent TB, the recognized assay to be include, in all works testing new methods, is the Quantiferon Gold as a reference assay. Authors must include the reason why they did not use it and discuss how this could affect the results they found. I mean, how would they compare how good is the method they propose against a recognized one. Also it would be good if they have had the quantiferon data for each patient for a better group distribution. They neither did it in the previous work in reference 9.

Two preparations were tested in this work: membrane-associated antigens and Acr protein. Authors suggest than membrane antigens could be a better marker than Acr for latent TB. However, the 82 antigens identified in the previous study as immunogenic were not analyzed for their specificity for Mtb. The question is if there are some cross reactivity with other mycobacteria species and that could confuse the results and make it harder to interpret them. Authors reported that antibodies and T cell response not being influenced by tuberculin or BCG vaccination, even so, it cannot be excluded a cross reactivity with membrane antigens. Also, it turns out that Acr is one of the main protein of the antigens associated to the membrane; then, how do you know that the response is due to other antigens or mainly due to Acr? A more extensive study about Mtb membrane antigens would give a better understanding to select the correct antigens for the assays.

Another emerging question is the number of samples, I think they are not enough to have right conclusions, but this is already mentioned by the authors in discussion.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-19-27417R1

Immune responses to Mycobacterium tuberculosis membrane-associated antigens including alpha crystallin can potentially discriminate between latent infection and active tuberculosis disease

PLOS ONE

Dear Dr Sinha,

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.

We would appreciate receiving your revised manuscript by Feb 17 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,

Katalin Andrea Wilkinson, PhD

Academic Editor

PLOS ONE

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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 #1: (No Response)

Reviewer #2: 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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: (No Response)

Reviewer #2: (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 #1: Yes

Reviewer #2: (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 #1: Yes

Reviewer #2: (No Response)

**********

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: The authors have undertaken substantial revision of the manuscript exploring humoral immunogenicity of membrane-associated proteins in human TB. The principle findings include (a) higher levels of IgM in contacts than in active TB patients (b) higher Acr IgA in active TB than contacts (c) altered ratios of IgG to Acr vs Mtm across the spectrum of TB disease.

In general, the comments has been addressed.

Further comments:

1. The major concern of statistical analysis has been resolved with the correction of non-parametric and multiple comparison tests.

2. The rebuttal to the flow cytometry gating is accepted. The live/dead stain at day 6 confirms the cells were still alive in active TB.

3. Thank you for the additional experiments regarding the antibody avidity. It is suggested to put the p-value in the figure given that the trend is discussed. Could the heterogeneity in antibody avidity in active TB be due to different durations of disease prior to presentation? Perhaps something to explore in future studies with greater powering.

4. The finding of IgM being greater in contacts than in active TB is interesting, suggesting the chronicity of active TB drives class-switching. Could a mechanism be included in the discussion?

5. The r-squared and p values should be inserted in the scatter plot figures (correlations).

6. The results regarding IgM not detectable against Acr is likely technical (concentration of Acr coating?), but the authors have addressed this clearly in the discussion. In the way it is presented, this could still be of benefit for the research community.

7. Line 268: Can immunoglobulin levels truly be directly compared on an OD basis given it is being compared across different antigens and secondary antibodies? Would this not require a standard curve of subclasses to derive a specific concentration of immunoglobulin? However, it could be correct to state that IgG and then later for figure 6, IgG2 (line 316), is the most readily detectable immunoglobulins in their given assays.

Minor comments:

1. Line 66: What is meant by ‘presumptive’ in single quotation marks?

2. Line 88: Missing a word – Mtb membrane lysate or proteins?

3. Line 97: Why is ‘Mtb complex’ in single quotation marks?

4. Line 95-96: What is meant by sustain the bacilli in

5. Line 188: Should be IgA not AgA

6. Line 200: The concentration of the secondary should be included.

7. Line 256: Missing word – elaborated ‘on’.

8. Line 394: It is unclear what is meant by quantum of infection increasing.

9. Line 556: Typo in semi-quantitative. The point with semi-quantitative is that levels of antibodies cannot be directly compared when using different reagents (secondaries) or assays. Further, it also poses a challenge for defining cut-off values for diagnostic tests as the units are arbitrary and contingent on the individual assay. However, it is accepted that this study is exploratory in nature.

10. Line 451: Why is dynamic in single quotation marks?

11. Line 492: Why is ‘cell-surface’ in single quotation marks?

12. Line 512: “gets worsened” should perhaps be “is worsened”

Reviewer #2: My comments about some of the responses given by authors

Author’s response: We have used TST as the reference assay, reasons for which have now been elaborated more explicitly in revised Introduction (2nd paragraph).

Authors have explained the reason why they used TST as the reference assay. I may consider this a valid reason, but it would be easier to compare their results with others reports using IGRAs. Still the data provided in this paper may be useful to complement the information about this topic.

Author’s response: We fully agree with this observation. In fact we have not proposed that the crude membrane should be used as such. In concluding paragraph of Discussion we had said that “In addition to Acr, the study draws attention to MtM as an emerging source of immunodominant T cell antigens”. We have now revised the Discussion to further clarify this point. However, in real life the ‘non-tuberculous mycobacteria’ as well as BCG do not seem to confound the results of TST which also uses a crude antigen (Farhat et al, Int J Tuberc Lung Dis, 2006; 10:1192).

I agree with the paragraph mentioned in discussion. But the fact that MtM is a rich source of antigens, most of them unstudied, reveals that it is not close to determine which protein may actually function as biomarker. As author also responded: we have made some such attempts in the past (ref 8) but have not been able meet any remarkable success.

Additional comment:

I expected to see the changes made to the paper highlighted to facilitate the second review, instead I had to go through all the paper to see the changes

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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.

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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 to be viewed.]

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Immune responses to Mycobacterium tuberculosis membrane-associated antigens including alpha crystallin can potentially discriminate between latent infection and active tuberculosis disease

PONE-D-19-27417R2

Dear Dr. Sinha,

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

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Katalin Andrea Wilkinson, PhD

Academic Editor

PLOS ONE

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