Immunoinformatics design of a novel multiepitope vaccine candidate against non-typhoidal salmonellosis caused by Salmonella Kentucky using outer membrane proteins A, C, and F

Elayoni E. Igomu; Paul H. Mamman; Jibril Adamu; Maryam Muhammad; Abubarkar O. Woziri; Manasa Y. Sugun; John A. Benshak; Kingsley C. Anyika; Rhoda Sam-Gyang; David O. Ehizibolo

doi:10.1371/journal.pone.0306200

Peer Review History

Original SubmissionJune 12, 2024
30 Jul 2024 Decision Letter - Prashant Sharma, Editor PONE-D-24-23783Immunoinformatics design of a novel multiepitope vaccine candidate against non-typhoidal salmonellosis caused by Salmonella Kentucky using outer membrane proteins A, C, and FPLOS ONE Dear Dr. Igomu, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== We have now received the reports from our reviewers, and after careful consideration, we have decided that your manuscript requires major revisions before it can be considered for publication. The reviewers have provided detailed feedback that we believe will significantly enhance the quality and clarity of your manuscript. Key areas requiring revision include: 1. Sequence Diversity: Provide detailed analysis and presentation of the sequence diversity of the outer membrane proteins (OmpA, OmpC, and OmpF). 2. Language and Structure: Address significant grammatical errors and improve the manuscript's clarity and organization. Consider using professional editing services. 3. Methodology: The methods section should include additional technical details and present a figure explaining the stepwise flow of the methodology. 4. Experimental Data: Discuss potential challenges and limitations of the immunoinformatic approach and provide a rationale for the chosen methodologies. Cite references that have used similar approaches with in vivo evaluations. Addressing these comments will significantly strengthen your manuscript. Please submit a revised version of your manuscript with a detailed response to each reviewer's comment. If you have any questions or need further clarification, please do not hesitate to contact us. ============================== Please submit your revised manuscript by Sep 13 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'. 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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, Prashant Sharma, Ph.D. Academic Editor PLOS ONE Journal requirements: 1. When submitting your revision, we need you to address these additional requirements. 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 note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. 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For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. [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: Partly Reviewer #2: Partly Reviewer #3: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A Reviewer #3: 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 Reviewer #3: 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 Reviewer #3: 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: The authors employed a bioinformatics approach to analyze the physicochemical properties of three outer membrane proteins of Salmonella Kentucky and subsequently predicted their antigenicity and immunogenicity. This method demonstrates an effective and predictive strategy for developing a pipeline for various vaccines. Furthermore, by considering the HLA types of different ethnic groups, the study assesses whether these targets can be used universally for vaccines or diagnostics. However, a significant concern is the lack of clarity regarding the sequence diversity of the outer membrane proteins (OmpA, OmpC, and OmpF) analyzed throughout the paper, which limits the efficacy of the proposed target regions. 1. The authors collected outer membrane sequences with 99% to 100% identity using BLAST, resulting in a narrow range of antigenic sequences that may not be suitable for protection against various Salmonella Kentucky strains. The authors should describe the number of "complete coding sequences (CDS)" as a percentage. 2. Additionally, the candidate regions cover variation of OmpA, OmpC, and OmpF in all Salmonella Kentucky strains? The diversity of OmpA, OmpC, and OmpF should be analyzed to understand the sequence diversity and efficacy of the vaccine/diagnostic candidates. To address this, a maximum likelihood phylogenetic tree based on codon alignments using a non-redundant CDS collection of the Omp genes should be presented. Authors should confirm whether clusters in the phylogenetic tree correlate with amino acid sequence variation in the candidate regions. Reviewer #2: The manuscript presents a substantial amount of valuable data that is highly relevant and can contribute significantly to the field of vaccinology. However, in my opinion the paper has some Shortcomings and the manuscript's current structure and clarity need significant improvement. Major grammatical errors present, the manuscript needs language editing. Consider using professional editing services. Method section needs to be more systematically written with sufficient technical details to be more detailed and clearly described. While the data is extensive, the results section is challenging to follow. Specific comments 1. The manuscript lacks proper punctuation, including brackets and full stops eg; pg 4, line 86, pg5, line 111 2. Pg4, line 90: what are the various risk factors? 3. Italicise all species names 4. Please ensure that capitalization is used correctly eg: pg6, line 128: Human 5. Good to have a figure explaining the stepwise flow of the methodology. 6. Use the right terminology, pg 6, line 113: BLOSUM62 is substitution matrix. 7. Was the signal peptide identified? 8. Pg 5, line 97: FASTA format sequences in aa or nucleotide? 9. Describe in detail the alleles used for T‑Cell epitope prediction analysis. 10. What is the IC50 threshold values set? 11. Pg 6, line 116: Epitope Analysis Resource from Immune Epitope Database (IEDB) server. 12. Why ab initio method was preferred for tertiary structure prediction? 13. Table 2 can be moved to supplementary file. 14. The number of OMP sequences selected varies, OMPF has only 15 sequences? 15. Table 3: can differentiate the epitopes by the protein (OMP A, C and F) 16. Table 6; positive (typo), differentiate the OMPs. 17. Resolution of the figures could be improved. 18. Figure legends/captions should include a clear and concise description of the figure. eg Figure 7: Docking of the 3D tertiary structural models (list the colours of the MEV and TLRs) Reviewer #3: PONE-D-24-23783 General comment: The research article "Immunoinformatics design of a novel multiepitope vaccine candidate against non-typhoidal salmonellosis caused by Salmonella Kentucky using outer membrane proteins A, C, and F". The research demonstrates a well-structured approach to vaccine design using bioinformatics and immunoinformatics techniques. The study created a novel MEV construct from OMP A, C, and F with the inclusion of fliC protein as an adjuvant from Salmonella typhimurium and characterized it using various software. The MEV was identified as having 14 CD8+ and 7 CD4+ T-cell epitopes, along with immunogenic B-cell epitopes. The vaccine construct demonstrated high structural quality, favorable physicochemical properties, and stability in molecular docking and dynamic simulations. Immune simulations indicated significant dose-dependent immune responses of IgG and IgM. The MEV is predicted to be non-toxigenic and non-reactogenic in nature. Specific comments: The authors used immunoinformatics tools to construct a potential MEV candidate for Salmonella Kentucky and characterized it with various software / in-silico tools to predict interferon-gamma (IFN-γ) 420 induction capabilities, discontinuous B-cell epitopes, secondary and tertiary structure, Protein-protein docking and protein-protein binding affinity, Molecular dynamics, and Immune Simulation. The authors also cited various reference (38, 47, 48, 54, 62, 63, 66, 73, 77, 78, 81, 82) which has used similar approach of immunoinformatics to design vaccine for various infectious diseases. However, the finding does not provide experimental data to support the theoretical findings, which is crucial for translating the research into a viable vaccine candidate. This study could benefit from a more detailed discussion of the potential challenges and limitations of this approach. Additionally, it is important to cite the reference who used the immunoinformatics approach to design a vaccine candidate and evaluated it in small animals to support in-silico Vs in-vivo results. Section 2.8: • Line no 320 - “Ensuring the efficacy of the vaccine model during cloning and expression is crucial for its in-vitro production” should be changed to “Ensuring the efficacy of the vaccine model during cloning and expression is crucial for its in-vivo production” • It is not clear why authors in this study performed codon optimization for a vaccine construct for a mammalian host. The MEV proposed is a bacterial-derived chimeric protein construct that can be easily expressed in a simple host like E. coli. This poses a question on the rationale for the construction of MEV codon. Section 2.19: • It is not clear why the authors introduced 6xHis tag on the C-terminal of the MEV in this study as all the experiments were in silico. It is recommended to avoid His tags in vaccine candidate when predicting their immune response using the database. The MEV containing His tag may potentially lead to the generation of anti-histamine antibodies. • Authors have also not described whether any antibiotic marker is used in a plasmid construct. Section 3.6 and discussion section: The authors have not described the rationale for the order of linkage B cell, HTL and CTL epitope peptide linkage in a MEV construct. Was it random or a particular order of different peptide sequences of epitopes were followed to construct MEV? Section 3.12 and discussion section: • The immune simulation predicted the humoral responses 565 for both IgM and IgG were more pronounced after booster doses compared to the initial dose, with IgM levels being higher than IgG (and its isotypes). A detailed discussion would be beneficial to describe the significance of a higher level of IgM as compared to IgG and what it means. • A detailed discussion on the rationale of three doses and dose intervals while performing an immune stimulation. Section 5.0: Line no. 732: The author claimed MEV to be thermostable. However, the rationale for this claim is neither mentioned in the result section nor described in the discussion section. What parameters or scores were responsible for predicting good solubility and thermos-stability? ****** 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: No Reviewer #3: Yes: Ruchirkumar Pansuriya ******** [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. 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Revision 1
6 Sep 2024 Author Response PONE-D-24-23783 Key areas addressed as requested by the academic editor 1. To better understand the antigenic diversity for the different classes of OMPs (OmpA, OmpC, and OmpF), the percentage conservation of the amino acid sequence has been computed using the Phylogeny.fr server (Section 2.2 line 121), the results are in section 3.1 and table 2. The phylogenetic relationships, based on the maximum likelihood method, have been computed as recommended (Fig 2). 2. The language and structure have been greatly improved as recommended. Several paragraphs have been restructured to enhance clarity, professional tone, and flow. Attention has been given to punctuation details, abbreviations, table headers, and legends. 3. A flow chart explaining the methods in a stepwise manner has been included in the manuscript as recommended (Fig 1). The methodologies were improved upon, and the rationale behind the methods used was stated. You can find them in sections 2.2 (Lines 121 to 129 ), 2.9 (lines 223 to 226), section 2.17 (Lines 333 to 344), section 2.18 (lines 349 to 354 ), and 2.19 (lines 376 to 383) 4. The potential challenges and limitations of the use of the immunoinformatics approach have been provided (Section 6.0). Finally, I have incorporated references to studies that have used an in-silico approach to vaccine design with in-vivo evaluations (References 18, 35, 47, 69, 70, and 71) Response to reviewers Response to Reviewer # 1 1. Outer membrane proteins (OMPs) are highly conserved and identical within each class of protein in Salmonella, regardless of the serovar (as stated in lines 80 to 83). However, the percentage conservation across different classes of amino acid sequences has been computed and presented in section 3.1 (lines 394 to 403) and Table 2. To avoid ambiguity, section 2.1 has been rephrased to clarify intent. The reason for blasting at 99 % to 100% for each class of OMP in our study was majorly to narrow down our selection criteria, to ensure all selected proteins were those isolated from Salmonella Kentucky. This does not in any way narrow the antigenic range within each class of protein. 2. To better understand the antigenic diversity for the different classes of OMPs (OmpA, OmpC, and OmpF), a phylogenetic tree based on maximum likelihood has been computed and added as recommended (Fig 2). Response to Reviewer # 2 1. Punctuations have been improved, typographical errors corrected and the right terminologies noted. 2. A statement on the various risk factors that make Africa prone to the emergence of AMR clones has been added (Lines 90 to 93). 3. All species names have been italicized 4. The IC50 threshold value chosen has been stated (Line 160). 5. Tables have been updated to differentiate epitopes by protein classes (Tables 3 and 6). 6. The use of “amino acid” in FASTA format has been clearly stated (Section 2.1) 7. Legends for figures 3, 4, 5, 6, and 12–16, have been updated to improve clarity and conciseness. 8. Figure 9, formerly Figure 7, depicting the dock complex between TLRs and MEV, has been updated to include the color coding between MEV and TLRs, as recommended. Response to Reviewer # 3 1. References to studies that used the immunoinformatics approach to design a vaccine candidate and evaluated it in small animals to support in-silico Vs in-vivo results have been included (18, 35, 47, 69, 70, 71). 2. The rationale for the choice of plasmid and the choice of dosing regimen has been explained in the manuscript as advised and references that have used similar approaches are cited (Section 2.17, Line 333–344; Section 2.18, line 349–355). References 39, 65, 66, 67, 68. 3. Codon optimization was performed for mammalian hosts because the vaccine was designed for humans. In vitro experiments (e.g., transfection studies) are often conducted in mammalian cell lines for human vaccines. Additionally, most in vivo experiments are performed in laboratory animals like mice, rats, and monkeys, which are mammals. Studies that employed mammalian codon optimization and or, the pcDNA3.1 plasmid, have been referenced in the manuscript (sections 2.18 and 2.19), specifically in references 18, 35, 68, 69, and 70. 3. The immune simulation study was done using the amino acid (AA) sequence of the multi-epitope vaccine construct without the 6xHis tag AA sequence. The inclusion of the 6xHis tag was during cloning into the plasmid after the immune simulation had been conducted. Studies with similar approaches have been referenced (Reference 47). The addition of the 6xhis tag to the protein before cloning was to make it easy to track the protein during expression and purification processes when conducting in-vitro experiments. The detection of anti-His antibodies is what makes it easier to track the proteins of interest during expression and purification processes. Finally, 6xHis tag usually does not interfere with the protein’s native structure or function. 4. Most in-silico designs employ plasmid with antibiotic markers. They are utilized to identify and successfully select transformants, particularly in cloning, gene expression, and genetic engineering. This study and other studies referenced in this manuscript employed plasmids that carry antibiotic markers. The AmpR (ampicillin-resistant) gene carried on our plasmid was the marker (Fig 18). 5. The rationale for the order of linkage of the epitopes for the construction of the MEV has been included in the manuscript (Section 2.9, lines 223–227). 6. A detailed discussion on the implication of the high IgM has been included in the manuscript (Lines 762 – 773). 7. The physicochemical properties of the vaccine that make it thermostable and soluble are available in the results (Section 3.7, Lines 488 to 490 for solubility; and lines 491 to 494 for aliphatic index). These parameters and scores have been quoted in the discussion (lines 735– 739). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0306200.r002
9 Oct 2024 Decision Letter - Prashant Sharma, Editor PONE-D-24-23783R1Immunoinformatics design of a novel multiepitope vaccine candidate against non-typhoidal salmonellosis caused by Salmonella Kentucky using outer membrane proteins A, C, and FPLOS ONE Dear Dr. Igomu, 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. Both reviewers have recommended minor revisions. Given this, I would kindly ask you to carefully review the manuscript for any areas that may benefit from further clarification or minor improvements, including correcting typographical errors, enhancing the explanation of key concepts, or addressing any formatting inconsistencies. Authors should ensure the text is as clear and polished as possible before resubmission. Please submit your revised manuscript by Nov 23 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. 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We look forward to receiving your revised manuscript. Kind regards, Prashant Sharma, Ph.D. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 #3: All comments have been addressed ******** 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: Partly Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #3: 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 #3: 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 #3: (No Response) ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: I understand the authors' point. My question is whether the vaccine candidate developed by the authors could also be effective against other strains of Salmonella Kentucky, which sequences are available in NCBI but were not discussed in this article. Reviewer #3: (No Response) ****** 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 #3: 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. Attachments Attachment Submitted filename: PONE-D-24-23783_Review_2-Ruchir Pansuriya.docx https://doi.org/10.1371/journal.pone.0306200.r003
Revision 2
4 Nov 2024 Author Response PONE-D-24-23783R1 Key areas addressed as requested by Journal I have carefully reviewed all references cited in this manuscript to confirm their accuracy and completeness and can confirm that no retracted articles have been cited or identified. Response to reviewers Response to Reviewer # 1 Yes. The developed vaccine candidate is expected to be effective against all strains of Salmonella Kentucky, including those with and without available sequences in the NCBI database, as the outer membrane protein sequences used in this study incorporate highly conserved regions. Response to Reviewer # 3 Response to Specific Comment (2.18 Codon Adaptation): The authors appreciate the insightful feedback regarding glycosylation concerns for the MEV construct expressed in mammalian cells. The authors recognize the potential for glycosylation at certain amino acid residues. Although the in-silico modeling did not integrate genetic interventions to address glycosylation, this issue can be effectively addressed during in-vitro experimentation: 1. Post-Expression Enzymatic Treatment: In-vitro assays can utilize PNGase F (N-Glycosidase F) to remove any N-linked glycans after expression enzymatically. While this does not inhibit glycosylation during protein synthesis, it ensures that the final product remains glycosylation-free, making it a closer structural analog to the bacterial protein. 2. Use of Glycosylation-Deficient Cell Lines: Certain cell lines have been genetically modified to lack glycosylation pathways, such as CHO-K1 cell lines, which we intend to select for this purpose. Using these glycosylation-deficient cell lines minimizes or eliminates unintended glycosylation during protein expression. Given that this manuscript focuses exclusively on in-silico findings, the authors have not detailed the use of CHO-K1 cell lines in this research phase. We anticipate that these in-vitro techniques will adequately address glycosylation concerns in future experimentation phases. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0306200.r004
7 Nov 2024 Decision Letter - Prashant Sharma, Editor Immunoinformatics design of a novel multiepitope vaccine candidate against non-typhoidal salmonellosis caused by Salmonella Kentucky using outer membrane proteins A, C, and F PONE-D-24-23783R2 Dear Dr. Igomu, 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, Prashant Sharma, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0306200.r005
Formally Accepted
11 Nov 2024 Acceptance Letter - Prashant Sharma, Editor PONE-D-24-23783R2 PLOS ONE Dear Dr. Igomu, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Prashant Sharma Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0306200.r006

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