A fusion protein comprising pneumococcal surface protein A and a pneumolysin derivate confers protection in a murine model of pneumococcal pneumonia

Tanila Wood dos Santos; Pedro Almeida Gonçalves; Dunia Rodriguez; José Aires Pereira; Carlos Augusto Real Martinez; Luciana C. C. Leite; Lucio F. C. Ferraz; Thiago Rojas Converso; Michelle Darrieux

doi:10.1371/journal.pone.0277304

Peer Review History

Original SubmissionJune 20, 2022
21 Sep 2022 Decision Letter - Ray Borrow, Editor PONE-D-22-17627A fusion protein comprising Pneumococcal surface protein A and a Pneumolysin derivate confers protection in a murine model of pneumococcal pneumonia.PLOS ONE Dear Dr. Darrieux, 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 below during the review process. Please submit your revised manuscript by Nov 05 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. 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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: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No ****** 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: Overall this is a reasonable manuscript that follows up on a previous study. For the most part, the data supports the conclusions. Two points should be addressed. 1. While the purpose was to examine protection against pneumonia as opposed to sepsis, why was this data not included in the sepsis manuscript. There have already been pneumonia challenge models described for the pneumococcus. The authors could have use one of those rather than develop their own. 2. The authors state that their model had pneumococci restricted to the lungs and no other organs. However, there is no data to support this clam. Reviewer #2: Summary observations Abstract is clear and so is the introduction. The authors demonstrate the scientific basis for initiating the study. Appropriate references are quoted, institutional review board’s approval was obtained. The materials and methods section (mainly the Mouse pneumonia model part) need needs to be modified to reflect what is presented in the results section (Murine Model of pneumonia) (see Specific comments). The data were collected, analyzed, and interpreted by and large correctly. Figures are clear and readable but there are mistakes in some of the Figures. The results are adequately discussed. Specific Comments Introduction L60 and L74 may have to be rewritten to avoid starting a new paragraph with a backward link to the previous paragraph but to start with a fresh topic sentence. Where there is a backward link, readers may conclude that this is simply ‘more of the same’ and so skip onwards to the next paragraph. Materials and Methods L89 and L126 ‘St’ is written in italic while elsewhere it is not (see L144, L147, L153. L199 and L246). In L177 it is ‘Sp’. L144-147(Results section) and L254-258 - the authors present and discuss ‘lung colonization and determination of colonization end-point’; 5 days versus 7 days. However, information about comparing St 245/00 and P854 colonization of the lung (5 days versus 7 days) and determination of the endpoint is missing in the materials and methods section (mouse and pneumonia model). In fact, L111 starts with the words ‘After 7 days’ giving the impression that the comparison was done for ‘day 7’ only. L116 - “Comparison between groups was performed using Students t test’. It is not clear which groups the authors are referring to. It is confusing. Is it BALF verses Lung? Or St 245/00 versus P854. Clarify. Results L114 (Methods-Mouse pneumonia model) reads ‘Serial dilutions of BALF…………were plated on blood agar…. for determination of bacterial counts …’. However, no data on BALF bacterial counts are provided; only CFU in the lung in presented (see Figure 1A) In Figure 1A, the authors used ‘Lung CFU’ (Y-axis label) and in Figures 2A and 2B used ‘CFU in BALF’ and ‘CFU in Lung’ respectively. For consistency’s sake use the same ‘style’ of labelling. L126 - L128. BALF was collected after seven days to determine cellular infiltrate and cytokine production. However, Figures 3A and 4A &B are for cellular infiltrate and cytokine production for time points 2-48 hrs and not for day 7. Clarify the ‘‘After seven days.’ L129 reads ‘Blood and liver were also collected for CFU count’. No data are provided. L146 -L147 read ‘…we have proceeded with the challenge experiments using St 245/00 and 7 days as the colonization endpoint.’ and L255 goes ‘Challenge with the serotype 14 strain, St 245/00, resulted in lung colonization at day five, which increased at day seven, with less variations among individuals. Therefore, this strain was chosen to evaluate the effects of vaccination on pneumonia.’ Given that both St 245/00 and P854 gave ‘comparable’ results (Figure 1A), it would appear then that St245/00 was chosen on the basis of ‘less variations among individuals., However, the data on ‘variations among individuals’ have not been provided. L168-L169 and L178 refer to bacterial loads in the BALF but Figure 2B is about ‘CFU in Lung” Figure 1B-The Y axis label should be ‘BALF cells x 104/ml’ not just ‘cells x 104/ml’ Figure 4A and B. In the legend, it should be 48 hrs not 168hrs ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: Enoch Sepako, PhD ******** [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.0277304.r001
Revision 1
11 Oct 2022 Author Response We would like to thank the reviewers for the careful analysis of the manuscript. All the suggestions were accepted and incorporated in the final version (the modifications are highlighted in yellow). We have addressed each comment individually, as follows: Reviewer #1: Overall this is a reasonable manuscript that follows up on a previous study. For the most part, the data supports the conclusions. Two points should be addressed. 1. While the purpose was to examine protection against pneumonia as opposed to sepsis, why was this data not included in the sepsis manuscript. There have already been pneumonia challenge models described for the pneumococcus. The authors could have use one of those rather than develop their own. 2. The authors state that their model had pneumococci restricted to the lungs and no other organs. However, there is no data to support this clam. 1. The sepsis data was generated earlier as part of the project of another student at the lab. She was comparing different vaccine formulations including Pspa and pneumolysoids, and she focused on the sepsis model, which was already well stablished in our lab. The pneumonia model was stablished later, when we identified pneumococcal strains of serotypes with lower invasiveness to use in this infection model. We have, in fact, based our pneumonia model in previously described work, but we selected strains from our bacterial bank and therefore some adjustments were necessary due to particularities in these specific strains. 2. No bacteria were found in the blood and liver of the immunized or control mice in any time point after challenge. This information was added to the text (lines 170-171). Reviewer #2: Summary observations Abstract is clear and so is the introduction. The authors demonstrate the scientific basis for initiating the study. Appropriate references are quoted, institutional review board’s approval was obtained. The materials and methods section (mainly the Mouse pneumonia model part) need needs to be modified to reflect what is presented in the results section (Murine Model of pneumonia) (see Specific comments). The data were collected, analyzed, and interpreted by and large correctly. Figures are clear and readable but there are mistakes in some of the Figures. The results are adequately discussed. Specific Comments Introduction L60 and L74 may have to be rewritten to avoid starting a new paragraph with a backward link to the previous paragraph but to start with a fresh topic sentence. Where there is a backward link, readers may conclude that this is simply ‘more of the same’ and so skip onwards to the next paragraph. These sentences have been modified as suggested Materials and Methods L89 and L126 ‘St’ is written in italic while elsewhere it is not (see L144, L147, L153. L199 and L246). In L177 it is ‘Sp’. The abbreviations have been corrected and kept as St throughout the manuscript. L144-147(Results section) and L254-258 - the authors present and discuss ‘lung colonization and determination of colonization end-point’; 5 days versus 7 days. However, information about comparing St 245/00 and P854 colonization of the lung (5 days versus 7 days) and determination of the endpoint is missing in the materials and methods section (mouse and pneumonia model). In fact, L111 starts with the words ‘After 7 days’ giving the impression that the comparison was done for ‘day 7’ only. The section was corrected as indicated. L116 - “Comparison between groups was performed using Students t test’. It is not clear which groups the authors are referring to. It is confusing. Is it BALF verses Lung? Or St 245/00 versus P854. Clarify. The comparison was performed between days 5 and 7 of infection with each strain. This information was added to the manuscript. Results L114 (Methods-Mouse pneumonia model) reads ‘Serial dilutions of BALF…………were plated on blood agar…. for determination of bacterial counts …’. However, no data on BALF bacterial counts are provided; only CFU in the lung in presented (see Figure 1A) The reviewer is correct. For bacterial counts, we have collected and plated the lung homogenates at days 5 and 7 post-infection. We used the BALF for analysis of the cellular infiltrates at 0, 3, 6, 12, 24 and 48 h following challenge. This information was corrected in the methods section. In Figure 1A, the authors used ‘Lung CFU’ (Y-axis label) and in Figures 2A and 2B used ‘CFU in BALF’ and ‘CFU in Lung’ respectively. For consistency’s sake use the same ‘style’ of labelling. The figure has been adjusted as indicated. L126 - L128. BALF was collected after seven days to determine cellular infiltrate and cytokine production. However, Figures 3A and 4A &B are for cellular infiltrate and cytokine production for time points 2-48 hrs and not for day 7. Clarify the ‘‘After seven days.’ The information was corrected on the text. The animals were euthanized at various time points. L129 reads ‘Blood and liver were also collected for CFU count’. No data are provided. No bacteria were found in the blood and liver of the immunized or control mice in any time point after challenge. This information was added to the text (lines 170-171). L146 -L147 read ‘…we have proceeded with the challenge experiments using St 245/00 and 7 days as the colonization endpoint.’ and L255 goes ‘Challenge with the serotype 14 strain, St 245/00, resulted in lung colonization at day five, which increased at day seven, with less variations among individuals. Therefore, this strain was chosen to evaluate the effects of vaccination on pneumonia.’ Given that both St 245/00 and P854 gave ‘comparable’ results (Figure 1A), it would appear then that St245/00 was chosen on the basis of ‘less variations among individuals., However, the data on ‘variations among individuals’ have not been provided. Although both bacteria behaved similarly in the challenge experiment, the results were a little less disperse with St 245/00, as indicated by the slightly lower standard deviation bar on this group. This information was added to the manuscript (lines 143-145). L168-L169 and L178 refer to bacterial loads in the BALF but Figure 2B is about ‘CFU in Lung” The information was corrected in the text. Figure 1B-The Y axis label should be ‘BALF cells x 104/ml’ not just ‘cells x 104/ml’ Figure 1B has been adjusted as indicated. Figure 4A and B. In the legend, it should be 48 hrs not 168hrs The figure legends have been Attachments Attachment Submitted filename: Response to reviewer.docx https://doi.org/10.1371/journal.pone.0277304.r002
25 Oct 2022 Decision Letter - Ray Borrow, Editor A fusion protein comprising Pneumococcal surface protein A and a Pneumolysin derivate confers protection in a murine model of pneumococcal pneumonia. PONE-D-22-17627R1 Dear Dr. Darrieux, 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, Ray Borrow, Ph.D., FRCPath Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0277304.r003
Formally Accepted
2 Nov 2022 Acceptance Letter - Ray Borrow, Editor PONE-D-22-17627R1 A fusion protein comprising Pneumococcal surface protein A and a Pneumolysin derivate confers protection in a murine model of pneumococcal pneumonia Dear Dr. Darrieux: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Ray Borrow Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277304.r004

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