Dear Dr. Aslam,

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We look forward to receiving your revised manuscript.

Kind regards,

Syed Nisar Hussain Bukhari

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: No

Reviewer #2: I Don't Know

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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Reviewer #1: The study addresses an important global health problem and applies an immunoinformatics pipeline to design a multi-epitope vaccine candidate against Monkeypox virus. However, the current version requires substantial improvements before it can be considered for publication.

Major Comments:

1. Several similar computational vaccine studies for MPXV have already been published. Please clarify how this study differs and contributes new insights. A comparison with prior MPXV vaccine design papers would strengthen the work.

2. The rationale for selecting only the H3L protein should be better justified, with supporting evidence of its immunogenic role.

3. The manuscript does not provide sufficient details on prediction tools, databases, thresholds, and simulation parameters. For reproducibility, please include exact versions, cutoffs, and settings.

4. Claims of stability and strong binding must be supported with quantitative data (RMSD, RMSF, hydrogen bond numbers, binding free energies). Figures and tables should be included.

5. Please provide quantitative readouts (antibody titers, cytokine levels) rather than qualitative statements.

6. Clarify that cloning was performed computationally. Include codon optimization indices and GC content with visuals.

7. The conclusion should more explicitly state that the study is an in silico prediction only and requires experimental validation.

Minor Comments:

1. The abstract should include quantitative results rather than vague phrases.

2. Correct the keyword “immunoinformatic” to “immunoinformatics.”

3. Improve grammar (e.g., “has emphasize” → “has emphasized”).

4. Ensure recent references (2022–2024) on MPXV vaccine design are cited.

Overall: The manuscript has potential but needs major revision to meet PLOS ONE’s standards of methodological rigor, reproducibility, and clarity.

Reviewer #2: This article is solid in context and contributes valuable insights; however, several areas require refinement to enhance clarity and precision.

Abstract: The reference to 92.22% needs to be expressed more clearly. It is not immediately evident what “worldwide” refers to in this context. Please clarify.

Methods: It is unclear whether the cloning and testing were performed directly by the authors or whether these steps refer to previously published work. This distinction should be explicitly clarified to avoid ambiguity.

Results vs. Methods: Some descriptions in the Results section overlap with methodological details (e.g., the subsection on MHC Class II epitope prediction).

Numerical Consistency: The manuscript would benefit from consistent rounding of numerical values, ideally to two or three decimal places, to improve accuracy and readability.

Conclusion: The current conclusion is overly general. A more specific articulation of the study’s contribution and implications is needed. For example, highlight how the findings advance vaccine design, what novel contributions were made compared to existing approaches, and what the next steps for validation should be.

Overall: This is a complex and ambitious research study.

Ethical considerations: The study appears to have been conducted using computer-aided vaccine prediction methods. No major concerns were identified regarding dual publication, research ethics, or publication integrity.

The authors employed multiple comparative approaches, simulations, and iterative refinements to assemble a final predictive vaccine model. While this breadth adds value, it also contributes to the manuscript’s length. Rationalising text and focusing on the most critical findings could improve readability and impact.

Reviewer #3: The study addresses an important topic and follows standard computational pipeline. It has several strengths including:

- The topic is highly relevant given the ongoing concerns about Monkeypox virus outbreaks.

- The manuscript is well structured and covers the full computational pipeline.

- The choice of the H3L protein is reasonable and supported by prior literature.

However, it has certain areas for improvement including:

- The authors study should clarify what is new compared to other recent MPXV vaccine design papers using computational approaches.

- The manuscript requires editing for grammar and clarity to improve readability.

- The discussion and conclusion should place stronger emphasis on the limitations of in silico approaches.

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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: Dr Isse Ali

Reviewer #3: No

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Multi-epitope vaccine design against Monkeypox virus: An immunoinformatic approach

PONE-D-25-35744R1

Dear Dr. Aslam,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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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,

Syed Nisar Hussain Bukhari

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: The authors have addressed the review comments precisely, and this is a solid paper. The developed vaccine demonstrates broad global coverage based on detailed predictive models supported by appropriate statistical methods, and it shows strong conservation across a range of MPXV strains.

Reviewer #3: - The novelty was duly clarified.

- Grammatical precision issue was addressed.

- Limitations of in-silico approach were mentioned.

All the issues were hence addressed.

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: Yes: Dr Isse Ali

Reviewer #3: No

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