In silico design and immunoinformatics analysis of a universal multi-epitope vaccine against monkeypox virus

Samira Sanami; Shahin Nazarian; Sajjad Ahmad; Elham Raeisi; Muhammad Tahir ul Qamar; Shahram Tahmasebian; Hamidreza Pazoki-Toroudi; Maryam Fazeli; Mahdi Ghatreh Samani

doi:10.1371/journal.pone.0286224

Peer Review History

Original SubmissionMarch 24, 2023
18 Apr 2023 Decision Letter - Sheikh Arslan Sehgal, Editor PONE-D-23-08627In silico design and immunoinformatics analysis of a universal multi-epitope vaccine against monkeypox virusPLOS ONE Dear Dr. Ghatreh Samani, 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 Jun 02 2023 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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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. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: https://assets.cureus.com/uploads/review_article/pdf/102530/20220810-30968-1qo1gw.pdf https://www.researchgate.net/publication/345757265_Monkeypox_Virus_in_Nigeria_Infection_Biology_Epidemiology_and_Evolution In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. 3. Thank you for stating the following financial disclosure: "This study was financially supported by the Research Deputy of Shahrekord University of Medical Sciences with grant number 6411." Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. Please note that funding information should not appear in any section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript. "Funding This study was financially supported by the Research Deputy of Shahrekord University of Medical Sciences with grant number 6411." 5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 6. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. 7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [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: N/A ****** 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: 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: This research study is very interesting "In silico design and immunoinformatics analysis of a universal multi-epitope vaccine against monkeypox virus", here author tries to determine the structure of epitope based vaccine designing against monkeypox virus . Following comments need to be incorporated in the MS 1. The introduction section is weak in terms of referring to the environmental aspects of Monkeypox virus, https://doi.org/10.1515/reveh-2022-0221 should be used to strengthen this section. 2. The authors chose the residues forming the disulfide bond based on a χ3 angle between -87° and +97° and an energy value less than 2.2 kcal/mol. Do you have a reference for these values? If you have a reference, be sure to give that cite. 3. In section 2.2. give the reason for evaluating epitopes for inducing the production of IFN-γ and IL-4. 4. Accession numbers of TLR4 and cholera toxin B subunit should be mentioned in the MS. 5. Authors should explain why they used cholera toxin B subunit as an adjuvant by citing the source. Reviewer #2: The authors have used immunoinformatic approaches and designed a multi-epitope vaccine against monkeypox virus using the B and T-cell epitopes of the cell surface-binding protein and the envelope protein A28 homolog which has significance in the present research field. The authors have generated lots of data, but the manuscript is not well written. However, I would recommend major revision of this manuscript. Comments 1. The abstract section is not well written. The abstract section needs more improvement with mentioning the specific finding (such as how may T-cell and B-cell epitopes etc.) were used for the vaccine construct instead of providing the overall findings. 2. The main limitation of this study is the data size. The authors have used only 10 sequences for the cell surface-binding protein and only 5 sequences for the envelope protein A28 homolog. The authors could use more sequences because the authors have depended on the conserved regions of these proteins. There are 100 of sequences are available in the protein sequence database such as Uniprot or in NCBI protein database. It is not clear why authors used only limited numbers of sequences among the large number of sequences? 3. Since this monkey pox virus is spreading in multiple countries, the analysis and showing the population coverage for the identified T-cell epitopes was important which is missing in this important study. 4. The authors could use 2 different servers for the allergenicity prediction for the cross validation of the allergenicity of the predicted epitopes. 5. The authors have mentioned about the in silico cloning in the materials and methods section, but they have not shown the Multiple cloning site in Figure 11 which needs correction and reanalysis of the in silico cloning for this vaccine construct. 6. The authors have predicted the three-dimensional structure of the final vaccine construct, so why did they predict the secondary structural features of the vaccine construct. Please provide a strong rational of using these or I will suggest removing this section from the manuscript. 7. The authors predicted the discontinuous B-cell epitopes from the Vaccine construct, but they have not discussed the significance of these findings. 8. The authors have performed the MDS for the TLR-4 and Vaccine construct separately (Figure 10), but it was important to perform the MDS for the TLR4-vaccine complex for the checking the stability of the complex. The authors could perform a 100ns MDS instead of the 40ns to see the proper stability of the vaccine, TLR4 or the TLR4-vaccine complex. 9. I would say the discussion section needs more improvement. In last few months, there are several research articles have been published for the multi-epitope vaccine design using immunoinformatic approaches in different journals. Could you please explain why this study is unique in comparison with the published articles. 10. Figure legends need to be more descriptive. ****** 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: Utpal Kumar Adhikari ******** [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.0286224.r001
Revision 1
9 May 2023 Author Response The authors of this manuscript thank the editor and reviewers for making suggestions and recommendations that helped us to improve the quality of the manuscript. We have made the changes suggested by the reviewers. Journal 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 Ans: Thanks for the comments. All items were checked and corrected based on the PLOS ONE style templates. 2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: https://assets.cureus.com/uploads/review_article/pdf/102530/20220810-30968-1qo1gw.pdf https://www.researchgate.net/publication/345757265_Monkeypox_Virus_in_Nigeria_Infection_Biology_Epidemiology_and_Evolution In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. Ans: Thanks a lot for your attention. The previous publications were addressed (reference 5 in line # 49 and reference 14 in line #73). 3. Thank you for stating the following financial disclosure: "This study was financially supported by the Research Deputy of Shahrekord University of Medical Sciences with grant number 6411." Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Ans: Dear editor, the above item was done accordingly. 4. Please note that funding information should not appear in any section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript. "Funding This study was financially supported by the Research Deputy of Shahrekord University of Medical Sciences with grant number 6411." Ans: Dear editor, the above item was done accordingly. 5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ Ans: Dear editor, the above item was done accordingly. 6. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. Ans: It's done (line #268-270). 7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Ans: Thank you so much for your nice comment. It's done (line #962-978). Reviewer #1: 1. The introduction section is weak in terms of referring to the environmental aspects of Monkeypox virus, https://doi.org/10.1515/reveh-2022-0221 should be used to strengthen this section. Ans: As per your valuable comment , this has been included in the manuscript (line #58-59 and reference 9). 2. The authors chose the residues forming the disulfide bond based on a χ3 angle between -87° and +97° and an energy value less than 2.2 kcal/mol. Do you have a reference for these values? If you have a reference, be sure to give that cite. Ans: It was done accordingly (line #378-380 and reference 66). 3. In section 2.2. give the reason for evaluating epitopes for inducing the production of IFN-γ and IL-4. Ans: Thanks for the comments. It was done (line #156-158 and reference 47). 4. Accession numbers of TLR4 and cholera toxin B subunit should be mentioned in the MS. Ans: Thanks a lot for the attention. It was done (line #170 and line #231). 5. Authors should explain why they used cholera toxin B subunit as an adjuvant by citing the source. Ans: As per your valuable comment , this has been included in the manuscript (line #531-534 and reference 108-111). Reviewer #2: 1. The abstract section is not well written. The abstract section needs more improvement with mentioning the specific finding (such as how may T-cell and B-cell epitopes etc.) were used for the vaccine construct instead of providing the overall findings. Ans: Thank you so much for your nice comment. The abstract of the paper was thoroughly revised accordingly as the reviewer comment. 2. The main limitation of this study is the data size. The authors have used only 10 sequences for the cell surface-binding protein and only 5 sequences for the envelope protein A28 homolog. The authors could use more sequences because the authors have depended on the conserved regions of these proteins. There are 100 of sequences are available in the protein sequence database such as Uniprot or in NCBI protein database. It is not clear why authors used only limited numbers of sequences among the large number of sequences? Ans: Thanks a lot for the attention. When we wrote this article, only these sequences were submitted and we only used them, according to your opinion, we also took the new sequences and analyzed them, and revised this part (line #120-124, line # 274-275). 3. Since this monkey pox virus is spreading in multiple countries, the analysis and showing the population coverage for the identified T-cell epitopes was important which is missing in this important study. Ans: Thank you so much for your constructive comment. this has been included in the manuscript (line #173-178, line #310-318, line #545-546). 4. The authors could use 2 different servers for the allergenicity prediction for the cross validation of the allergenicity of the predicted epitopes. Ans: Thank you for your valuable comment. If we use another server, we will lose some epitopes, and since the vaccine construct is designed, we cannot do this in this study, but we will definitely consider it in the next works. However, in many published articles, such as the articles below, one server has been used to determine the allergenicity of epitopes. DOI: 10.3390/vaccines10050691 DOI: 10.1038/s41598-022-12651-1 DOI: 10.3390/ijerph19095568 DOI: 10.3390/vaccines10081300 DOI: 10.1038/s41598-022-14427-z DOI: 10.1038/s41598-023-30445-x DOI: 10.1016/j.virusres.2020.198082 5. The authors have mentioned about the in silico cloning in the materials and methods section, but they have not shown the Multiple cloning site in Figure 11 which needs correction and reanalysis of the in silico cloning for this vaccine construct. Ans: This part and Figure 11 were revised based on the reviewer's comment, and the Multiple cloning site was marked in blue color in Figure 11 (line #257-266, line # 477-480). 6. The authors have predicted the three-dimensional structure of the final vaccine construct, so why did they predict the secondary structural features of the vaccine construct. Please provide a strong rational of using these or I will suggest removing this section from the manuscript. Ans: Based on the reviewer's suggestion, this section was removed from the manuscript. 7. The authors predicted the discontinuous B-cell epitopes from the Vaccine construct, but they have not discussed the significance of these findings. Ans: As per your valuable comment the above item were provided accordingly in the manuscript (line #576-580). 8. The authors have performed the MDS for the TLR-4 and Vaccine construct separately (Figure 10), but it was important to perform the MDS for the TLR4-vaccine complex for the checking the stability of the complex. The authors could perform a 100ns MDS instead of the 40ns to see the proper stability of the vaccine, TLR4 or the TLR4-vaccine complex. Ans: Thanks for the your valuable comments. We investigated the behavior of the vaccine and the receptor in the docked complex, but their graphs were drawn separately and in some studies, it was done in the same way (DOI: 10.3390/vaccines10122010, DOI: 10.1038/s41598-022-11851-z, DOI: 10.1016/j.ijbiomac.2020.07.117,DOI: 10.3389/fimmu.2022.865180, DOI: 10.1016/j.sjbs.2021.06.082), , but your comment is very interesting and constructive and will definitely be considered in future studies. After 33 ns, the vaccine reached an almost steady state and TLR4 also fluctuates very little during the simulation period. Due to the lack of facilities, this part of the work was done by a bioinformatics company, and due to financial limitations, we could only pay the company for 40 ns. I am sure you are aware of the bad financial situation of my country (Iran) and I hope you understand me. In many studies, such as the following studies, the simulation time is considered to be less than 40 ns (even 10 ns). DOI: 10.1038/s41598-022-14427-z DOI: 10.1021/acsomega.9b00944 DOI: 10.1007/s12026-022-09346-0 DOI: 10.1016/j.micpath.2020.104236 DOI: 10.1016/j.imu.2020.100478 DOI: 10.1371/journal.pone.0244176 DOI: 10.3390/vaccines10091564 9. I would say the discussion section needs more improvement. In last few months, there are several research articles have been published for the multi-epitope vaccine design using immunoinformatic approaches in different journals. Could you please explain why this study is unique in comparison with the published articles. Ans: Thank you so much for your constructive comment. This part was added to the discussion section(line #505-522, line # 545-547, line # 560-562, line # 566-569). 10. Figure legends need to be more descriptive. Ans: Based on your valuable recommendation, Figure legends were modified. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0286224.r002
11 May 2023 Decision Letter - Sheikh Arslan Sehgal, Editor In silico design and immunoinformatics analysis of a universal multi-epitope vaccine against monkeypox virus PONE-D-23-08627R1 Dear Dr. Ghatreh Samani, 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, Sheikh Arslan Sehgal, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0286224.r003
Formally Accepted
15 May 2023 Acceptance Letter - Sheikh Arslan Sehgal, Editor PONE-D-23-08627R1 In silico design and immunoinformatics analysis of a universal multi-epitope vaccine against monkeypox virus Dear Dr. Ghatreh Samani: 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 Dr Sheikh Arslan Sehgal Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0286224.r004

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