PONE-D-20-23751

Immune alterations in subacute sclerosing panencephalitis reflect an incompetent response to eliminate the measles virus

PLOS ONE

Dear Dr. Saruhan-Direskeneli,

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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Edgar Meinl, M.D.

Academic Editor

PLOS ONE

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i) what type of consent you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed), and

ii) whether informed consent was also obtained from adults in the study.

In addition, we note that you refer to patients as "boys" and "girls" however some patients are not minors. Please revise your manuscript to refer to these patients as "male" and "female".

Also, please clarify whether this study is prospective or retrospective. If this study was retrospective, please include the date(s) on which you accessed the databases or records to obtain the data used in your study. If the study was prospective, please describe the recruitment methods used.

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To comply with PLOS ONE submission guidelines, in your Methods section, please provide a more detailed description of your methodology, specifically:

a) provide more detailed criteria for the diagnosis of SSPE

b) provide the source, name, and catalog numbers of the ELISA kits for detecting measles

c) provide the source, catalog numbers, and dilutions for all primary/secondary/isotype control antibodies used in the study.

We look forward to hearing from you.

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This study is supported by Istanbul University Research Fund.'

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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

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

Reviewer #1: Yes

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 investigated the immune response in patients with SSPE in comparison to two well age-matched control groups consisting of inflammatory (ICON) and non-inflammatory (NICON) diseases. Taking into account that SSPE is a rare disease the number of patients is very high.

The authors used established methods (FACS, ELISA, Elispot assays, 3H thymidine incorporation). PBMCs of patients and controls were investigated ex vivo or after different stimulations in vitro – unspecific (antibodies, SAC) or MeV specific (viral peptides).

The results demonstrate, that PBMC of SSPE patients produced lower levels of IL-10 and IFN-g after stimulation with anti-CD3 and anti CD28, but were inducible to produce IL-2. After SAC stimulation PBMC of SSPE patients showed reduced IL-12p70 production and CD 14+ monocytes demonstrate lower CD46 surface expression. In Elispot assays spontaneous IFN-g production and antigen stimulated IFN-g production was elevated in SSPE patients and NICON compared with ICON.

The authors concluded, that T cells of SSPE patients demonstrate an altered immune response that is not sufficient to eliminate the virus. In monocytes reduced IL-12 production and CD46 surface expression implicate the effect of CD46 binding in SSPE similar to MeV infection.

Major points:

1. The authors report that in a recent epidemiological study of Istanbul a girl dominance was found. In their SSPE cohort there is a dominance of boys. Is this just by chance? From which part of the country or from which country were the patients recruited?

2. Different clinical stages of SSPS are known. For reference see e.g. Jabbour J, et al., SSPE-clinical staging, course, and frequency. Arch Neurol. 1975;32(7):493–494. 24 or Gutierrez J, et al., Dev Med Child Neurol. 2010 Oct;52(10):901-7. It would be interesting to know in which clinical state the patients had been at the time of blood sampling.

3. How do the authors interpret the reduction of T cells in SSPE samples?

4. The authors report “Monocyte stimulation with SAC”. In the legend of Fig. 4 they report “… SAC stimulated PBMC…”. If they don´t select monocytes before stimulation, the headline of this section and the legend of figure 4 should be modified. In addition, the authors should mention that SAC does not stimulate only monocytes in their cell culture.

5. At the beginning of the discussion the authors claim “… no evidence was found for immunosuppressive mechanisms as a determining factor in SSPE development.” The authors may explain more detailed the reasons for this statement or omit it.

6. The authors report a reduced production of IL-10 in the present study, but mention a production of IL-10 as in controls in their previous study. Do the authors have an explanation for this?

7. IL-10 is a cytokine with strong immunosuppressive properties. However, there are also publications demonstrating immunostimulation by IL-10, e.g. Il-10 enhance the capacity of resting CD4+ lymphocytes to produce cytokines. The authors may discuss this aspect as well and not solely the immunosuppressive properties of IL-10.

8. In the section “Antigen-specific T cell stimulation” the authors report “Interestingly, Ifn-g responses to all peptides and peptide pool were reduced in ICON group compared to SSPE (…) and NICON groups (…) …”. The authors may comment on this finding in the discussion, especially as they show an IFN-g response in PBMCs of ICON after unspecific stimulation (Fig. 2).

9. In the discussion the authors report “…SLAM expression was relatively higher in all cell subgroups both in SSPE and other inflammatory diseases compared with donors without inflammation…”. Fig. 3 demonstrate significant differences only for B-cells. This statement has to be modified.

10. The authors report that “… the donors have been difficult to assign to the respective groups.” Please explain the reasons.

Minor points:

1. Determination of CD46 and SLAM should be illustrated with an example showing a FACS analysis of a patient and two controls, e.g. as supplementary figures.

2. In the section “Antigen-specific T cell stimulation” there is a list of p-values. It is unclear to which peptide stimulation a given p-value belongs. The authors should present peptide stimulation and corresponding p-value in a supplementary table or omit the p-values in the text, as they are given in Fig. 6.

3. The citation format of two references in the discussion does not comply with the journal style.

Reviewer #2: In this study, authors aimed to test the cytokine profile of lymphocytes and monocytes obtained from the blood of SSPE patients to gain more insight into the immunopathogenesis of disease. SSPE is a latent brain infection that occurs many years after measles infection and it is a fatal disease. It is an important health problem in some of the developing countries. It is relatively frequent in Turkey, and a high number of SSPE patients were tested in this study. As SSPE is very rare, this study has a potential to contribute to the related literature. However, some points need to be cleared:

Minor revisions:

- Exclusion criteria for all groups should be mentioned.

- How would the authors interpret the finding that IL10 secretion is lower compared to controls? Is this a result of the latent brain infection, or is this more like a risk factor for the disease as a result of genetic factors etc? A comment on that in the discussion would be useful.

- What is the possible functional outcome of the finding that IL12 is decreased in monocytes? A comment on that in the discussion would be useful.

- The authors stated that “In SSPE patients, the lower frequencies ofCD46+ monocytes compared with NICON group in this study also implicated a related interaction of the virus with these cells.” However, SSPE is known as a latent brain infection and active involvement of monocytes in the periphery would be a surprising finding which may refer to ongoing MV activity in peripheral organs. Can the authors make this point clearer in discussion?

- In the conclusion, authors state that their findings pointed at an attenuated inflammatory pattern at a chronic phase of SSPE. Considering that SSPE is a brain-restricted latent infection, what may be the reason behind the attenuated inflammatory pattern seen in the peripheral blood cells? Discussion of this point would be useful.

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Reviewer #1: No

Reviewer #2: Yes: Atay Vural

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Attachments

Attachment

Submitted filename: Plos review.docx

PONE-D-20-23751R1

Immune alterations in subacute sclerosing panencephalitis reflect an incompetent response to eliminate the measles virus

PLOS ONE

Dear Dr. Saruhan-Direskeneli,

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 Jan 07 2021 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,

Edgar Meinl, M.D.

Academic Editor

PLOS ONE

[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 #1: All comments have been addressed

Reviewer #2: (No Response)

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: 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

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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: 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: The authors improved the paper in general by providing some corrections and further explanations. However, some points would still be better to be improved:

Point #1: If there were no exclusion criteria, were there any patients or controls who have a concomitant acute/chronic infection (other than SSPE), or autoimmune disorder at the time of blood sampling?

Points #2 and #3: The authors provided appropriate answers.

Point #4: The lower frequencies ofCD46+ monocytes compared with NICON group in the current study is an interesting finding and as the authors state “may implicate a related interaction of the virus with these cells.” Can the authors provide data regarding the presence or absence of MeV RNA in monocytes from SSPE patients?

Point #5:

In response to the point #5, the authors commented that “With the given data, SSPE is considered as a result of reactivation of a latent infection after many years. The RNA from the measles virus has been isolated in the brain, eyes, and spinal cord in patients with SSPE and persistence of MeV was confirmed in PBMC even after years…. During persistence, viral RNA in PBMCs and lymphoid tissue is detected in B, T lymphocytes and monocytes. Persistent RNA in lymphoid cells may contribute to immune response dysfunction by altering the ability of cells to proliferate in response to immune signaling…”

Reference #39 given as the source for this information is a review article and from its references it is seen that persistence of MeV was shown in Macaque Monkeys, HIV-infected children, or shortly after acute infection, but not after many years, or in children with SSPE. However, this is an important and interesting point. Could the authors provide more specific literature, or better, could they provide data regarding the presence or absence of MeV RNA in lymphocytes and also in monocytes, in SSPE children vs controls?

In addition, the authors stated that “An immune response is generated against the virus and a strong antiviral immune response is induced as evidenced by the unusually high levels of antibody in serum and CSF. But it is not effective in eliminating virus or controlling replication in the CNS.”

This is again an interesting and important point. According to the authors’ hypothesis, persistence of MeV and slow replication of the virus in the peripheral lymphoid organs and blood can be a source for the latent brain infection. And generation of a higher titer of anti-measles antibodies would show the presence of the viral persistence, but would be ineffective to eliminate the virus. Can the authors provide the specific literature showing the higher titer of anti-measles IgG in SSPE patients compared to controls? Or better, could they provide the data as an addition to the current study?

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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.

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.

Immune alterations in subacute sclerosing panencephalitis reflect an incompetent response to eliminate the measles virus

PONE-D-20-23751R2

Dear Dr. Saruhan-Direskeneli,

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,

Edgar Meinl, M.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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

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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

**********

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

Reviewer #2: 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 #2: Yes

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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: 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: (No Response)

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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.

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