Comprehensive analysis of early T cell responses to acute Zika Virus infection during the first epidemic in Bahia, Brazil

Assia Samri; Antonio Carlos Bandeira; Luana Leandro Gois; Carlos Gustavo Regis Silva; Alice Rousseau; Aurelien Corneau; Nadine Tarantino; Christopher Maucourant; Gabriel Andrade Nonato Queiroz; Vincent Vieillard; Hans Yssel; Gubio Soares Campos; Silvia Sardi; Brigitte Autran; Maria Fernanda Rios Grassi

doi:10.1371/journal.pone.0302684

Peer Review History

Original SubmissionNovember 27, 2023
25 Jan 2024 Decision Letter - José Ramos-Castañeda, Editor PONE-D-23-37988Comprehensive analysis of early T cell response to acute Zika Virus infection during the first epidemic in Bahia, BrazilPLOS ONE Dear Dr. Rios Grassi, 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 Mar 10 2024 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, José Ramos-Castañeda, M.Sc., 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. Please include a complete copy of PLOS’ questionnaire on inclusivity in global research in your revised manuscript. Our policy for research in this area aims to improve transparency in the reporting of research performed outside of researchers’ own country or community. The policy applies to researchers who have travelled to a different country to conduct research, research with Indigenous populations or their lands, and research on cultural artefacts. The questionnaire can also be requested at the journal’s discretion for any other submissions, even if these conditions are not met. Please find more information on the policy and a link to download a blank copy of the questionnaire here: https://journals.plos.org/plosone/s/best-practices-in-research-reporting. Please upload a completed version of your questionnaire as Supporting Information when you resubmit your manuscript. 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 5. Please include a copy of Table 1 and 2 which you refer to in your text on page 8 and 15. 6. We notice that your supplementary figures are uploaded with the file type 'Figure'. Please amend the file type to 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list. 7. Please upload a copy of Supporting Information Figure/Table/etc. Supplemental Table 1-4 which you refer to in your text on page 8, 9, 10 and 16. [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: Partly ******** 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: In recent years, significant efforts have been made to understand the mechanisms of the immune response involved in the control of acute viral infections. The ZIKA virus (ZV) causes an acute infection that induces mild symptoms and is self-limited. However, the infection can sometimes induce the Gillian-Barre syndrome and microcephalia in the newborn. Although both the innate and adaptive immune responses are involved in the recovery from the infection, a few questions remain regarding the precise mechanisms. Previous work showed that in acutely infected patients with ZV, there is a massive response of NKs producing large amounts of IFN-gamma, which could be involved in early infection control. This work aimed to determine whether the T cells, important producers of IFN-gamma, could also have an early response in acutely infected patients. The study was based on blood samples obtained within the first 7 days of the beginning of symptoms of acutely infected patients (29) and samples from the same patients 13-19 days after the resolution of symptoms. As controls, samples from healthy donors (11) and donors infected with Chikungunya virus and/or Dengue virus were used. Using mass cytometry analysis for different immunological markers, the authors found that both CD4 and CD8 T cells with memory markers could produce IFN-gamma early in the infection and that, in part, the production of IFN-gamma was associated with the phosphorylation of STAT-5 (pSTAT-5), a key transcriptional factor to produce IFN-gamma. On the other hand, only about 27-30 % of the samples tested showed specific production of IFN-gamma against ZV antigens, specifically against the NS proteins. The author concluded that the ZV infection induces early T cells that produce IFN-gamma which may contribute to the control of the infection. It is an interesting article that contributes to the basic knowledge of the immune response against ZV infection. The manuscript is clear and well-written. However, some issues need to be clarified. Comments: 1.- The study's main conclusion is that T cells have an early response producing IFN-gamma in patients infected with ZV, which may contribute to the control of the infection. However, the percentage of this T cell population is about 0.2%, which is low in an acute viral infection. 2.- Although pSTAT-5 is a key transcriptional factor for the induction of IFN-gamma, a significant number of samples in which the cells were producing IFN-gamma did not show pSTAT-5. How do the authors explain this fact? 3.- The description of figures 3 and 4 in the Results sections is unclear. This section needs to be rewritten for clarity. 4.- A very important question from this study is whether the T cells that secrete IFN-gamma “spontaneously” early during the acute infection are ZV antigens specific. Only about 30 % of the samples analyzed recognized ZV antigens. Does it mean that there is a high proportion of TCR-independent T-cell activation? If so, what could be the mechanism? Could it be TLR-dependent? A possible TCR-independent mechanism must be discussed in the manuscript. 5.- On the other hand, only class-I restricted ZV were analyzed. Incorporating proteins or class II-restricted peptides as antigens to the assays, the percentage of samples positive for ZV could be increased. It would be important to mention this point in the discussion. Reviewer #2: Although this could be a nice piece of work with state of the art technology, the authors undergo a number of experiments to show exactly what you would expect of a T cell-mediated immune response to a viral infection. Moreover, the authors fall short of some of the procedures mentioned in the Methods section, as they describe several procedures but they do not show the results of those experiments. For instance, they have a paragraph within the methods section that claim a search of T cell epitopes and peptides, and no results for that in the manuscript, only for the viral proteins that are the targets of the response. The only part where they mention response to a peptide is the one that elicits a cross-reactivity to Dengue virus. Table 1 is missing, which made difficult the reviewing of the manuscript. In short, the manuscript could be a good contribution if the authors do a more thorough analysis of their data. ****** 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: Yes: Fernando Esquivel-Guadarrama Reviewer #2: Yes: Jose Moreno ******** [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. https://doi.org/10.1371/journal.pone.0302684.r001
Revision 1
24 Feb 2024 Author Response Point-by-point responses to the reviewers’ comments: Manuscript: PONE-D-23-37988 We would like to thank the reviewers for careful and thorough reading of this manuscript and for their comments and constructive suggestions, which help to improve the quality of this manuscript. Reviewer's Responses to Questions Our responses for each point are below in bold. Reviewer #1: In recent years, significant efforts have been made to understand the mechanisms of the immune response involved in the control of acute viral infections. The ZIKA virus (ZV) causes an acute infection that induces mild symptoms and is self-limited. However, the infection can sometimes induce the Gillian-Barre syndrome and microcephalia in the newborn. Although both the innate and adaptive immune responses are involved in the recovery from the infection, a few questions remain regarding the precise mechanisms. Previous work showed that in acutely infected patients with ZV, there is a massive response of NKs producing large amounts of IFN-gamma, which could be involved in early infection control. This work aimed to determine whether the T cells, important producers of IFN-gamma, could also have an early response in acutely infected patients. The study was based on blood samples obtained within the first 7 days of the beginning of symptoms of acutely infected patients (29) and samples from the same patients 13-19 days after the resolution of symptoms. As controls, samples from healthy donors (11) and donors infected with Chikungunya virus and/or Dengue virus were used. Using mass cytometry analysis for different immunological markers, the authors found that both CD4 and CD8 T cells with memory markers could produce IFN-gamma early in the infection and that, in part, the production of IFN-gamma was associated with the phosphorylation of STAT-5 (pSTAT-5), a key transcriptional factor to produce IFN-gamma. On the other hand, only about 27-30 % of the samples tested showed specific production of IFN-gamma against ZV antigens, specifically against the NS proteins. The author concluded that the ZV infection induces early T cells that produce IFN-gamma, which may contribute to the control of the infection. It is an interesting article that contributes to the basic knowledge of the immune response against ZV infection. The manuscript is clear and well written. However, some issues need to be clarified. Response: We appreciate the positive feedback from the reviewer. Comments: Query 1.- The study's main conclusion is that T cells have an early response producing IFN-gamma in patients infected with ZV, which may contribute to the control of the infection. However, the percentage of this T cell population is about 0.2%, which is low in an acute viral infection. Response: It is true that the T cell population spontaneously producing IFN-� in ZIKV+ patients is only 0.26 and 0.21% for CD4 and CD8 T cells, respectively. However, these percentages are 10-fold higher than in healthy donors ones (0.02%) with highly significant p values of < 0.0001 for CD4 and p=0.0002 for CD8+ T cells, emphasizing the robustness of our results at this very early stage of viral infection. While it is true that these proportions may be low compared to antigen-specific cells in some acute infections, we do not claim that these cells are antigen-specific. Query 2.- Although pSTAT-5 is a key transcriptional factor for the induction of IFN-gamma, a significant number of samples in which the cells were producing IFN-gamma did not show pSTAT-5. How do the authors explain this fact? Response: We agree with the reviewer that we detected pSTAT5 in only one-third to one-half of CD4 and CD8 T cells producing IFN-�. We cannot exclude the possibility that much weaker pSTAT5 signaling might be present, but we only considered strongly pSTAT5-positive cells. Moreover, we could only focus on STAT-5 signaling, although STAT-5 is not the only transcription factor that induces IFN-�, especially STAT-1��which we did not evaluated. Therefore, we cannot exclude the possibility that other transcription factors are involved in this IFN-� induction. Query 3.- The description of figures 3 and 4 in the Results sections is unclear. This section needs to be rewritten for clarity. Response: We apologize for this. As suggested by the reviewer we have clarified in the text the description of the figures 3, 4 and Supplemental Figure 2A. 4.- A very important question from this study is whether the T cells that secrete IFN-gamma “spontaneously” early during the acute infection are ZV antigens specific. Only about 30 % of the samples analyzed recognized ZV antigens. Does it mean that there is a high proportion of TCR-independent T-cell activation? If so, what could be the mechanism? Could it be TLR-dependent? A possible TCR-independent mechanism must be discussed in the manuscript. Response: We agree with the reviewer that we detected few T cells specific for the ZIKV antigens in the peripheral blood and few ZIKV responders, while “spontaneous” IFN-� production was increased in all patients and therefore may not reflect an antigen-specific TCR-mediated event. However, we cannot exclude the possibility that some of the antigen-specific T cells had already migrated into the tissue and were therefore no longer detectable in the peripheral blood. It is also possible that these cells were not yet sufficiently amplified enough to be detectable in PBMC at the early time points at which we performed our analysis. In addition, as discussed below, our 15-mer approach may have favored MHC class I restricted T cells, and we may have underestimated MHC class II cells. However, we agree with the reviewer’s hypothesis that a high proportion of these IFN-�-producing T cells could result from TCR-independent activation of bystander T cells in the context of high overall activation, as measured by Ki67 expression, in this highly acute viral infection. We do not claim that the increased proportion of these circulating T cells spontaneously producing IFN-� is antigen-specific, and we have mentioned a possible TCR-independent mechanism in our conclusion. To clarify our interpretation, we have added the following sentence in the Discussion: “Alternatively, the high proportion of IFN-� -producing T cells could be due to a TCR-independent activation of bystander T cells in the context of high general activation, as measured by Ki67 expression, and in this highly acute viral infection.” 5.- On the other hand, only class-I restricted ZV were analyzed. Incorporating proteins or class II-restricted peptides as antigens to the assays, the percentage of samples positive for ZV could be increased. It would be important to mention this point in the discussion. Response: We thank the reviewer for his comment and agree that our approach to epitope prediction was to define class I epitopes. However, we have synthesized 15-mers peptides that are known to recognize not only class I epitopes, but also promiscuous class II epitopes restricted to T cells. We cannot exclude the possibility that a greater proportion of cells producing IFN-�, and in particular CD4 T cell-mediated responses, would have been detected if we had also predicted class II T cell epitopes, as the reviewer suggests. However, as mentioned in the manuscript, since we were severely limited by both the number of individuals tested and the amount of cells available, we prioritized the analysis of the Zika-specific T cell response to class I only. As suggested by the reviewer, we have added a sentence and a reference in the Discussion indicating that class I and class II T cell epitopes were tested using the same approach. We have included the following sentence in the Discussion: “Since we did not have enough cells to evaluate both class I and class II-restricted ZIKV-specific T cell responses in the present study, we prioritized the prediction of class I-restricted T cell epitopes, but the use of class II-restricted T cell epitopes would certainly have increased IFN-γ production by ZIKV-specific T cells, as suggested by Eickhoff et al. [50]. Nevertheless, the 15-mer size of our peptides also allows the detection of promiscuous MHC class II-restricted T cells.” Reviewer #2: Although this could be a nice piece of work with state of the art technology, the authors undergo a number of experiments to show exactly what you would expect of a T cell-mediated immune response to a viral infection. Moreover, the authors fall short of some of the procedures mentioned in the Methods section, as they describe several procedures but they do not show the results of those experiments. For instance, they have a paragraph within the methods section that claim a search of T cell epitopes and peptides, and no results for that in the manuscript, only for the viral proteins that are the targets of the response. The only part where they mention response to a peptide is the one that elicits a cross-reactivity to Dengue virus. Table 1 is missing, which made difficult the reviewing of the manuscript. In short, the manuscript could be a good contribution if the authors do a more thorough analysis of their data. Response: We appreciate the positive feedback from the reviewer. We thank the reviewer for his comment concerning the “search of T cell epitopes and peptides, and no results”. In fact, we had originally included a table with the details of this search for T- epitopes and peptides, but to simplify the paper, we decided not to include it. Your comment shows that this table is necessary, so we have added it. We are really sorry that you did not have access to Table 1, probably an error occurred during submission. The presence of this table would have made it easier for you to read the manuscript. Sorry again. Attachments Attachment Submitted filename: renamed_679ee.docx https://doi.org/10.1371/journal.pone.0302684.r002
8 Apr 2024 Decision Letter - José Ramos-Castañeda, Editor Comprehensive analysis of early T cell response to acute Zika Virus infection during the first epidemic in Bahia, Brazil PONE-D-23-37988R1 Dear Dr. Rios Grassi, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, José Ramos-Castañeda, M.Sc., Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): I thank the authors of this manuscript for their efforts to respond efficiently and punctually to the reviewers' comments. I consider that the manuscript has the merits to be published, although I suggest that the authors pay attention to a particular point mentioned by one of the reviewers that involves a simple correction in the sentence. 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) ******** 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: Yes Reviewer #2: N/A ****** 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 ****** 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: I am satisfied with the answers to the questions asked. The only observation I have is that the following paragraph is confusing: "compared to HD (Figure 3). With respect to coexpression of activation markers, the proportion. In contrast the frequencies of CD45RACCR7+CD27+central-memory (TCM), CD45RA-CCR7-CD27+ transitional-memory" Also, the supplemental figure 2A is not referred to in the text. Otherwise, I consider that the manuscript can be published in its present form. Reviewer #2: The manuscript is, undoubtedly, interesting and its data are sound. I insist that the authors did not dig deeply enough into their results that deserve a much longer discussion. Nevertheless, it can be published without any problem. I hope they examine their data in detail again, particularly the information on CD4 T cells, that is barely commented, and publish a review on the topic. I am open to discuss with them anything on that regard. ****** 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: Yes: FERNANDO ESQUIVEL-GUADARRAMA Reviewer #2: Yes: José Moreno ******** https://doi.org/10.1371/journal.pone.0302684.r003

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .