PONE-D-24-18487Efficacy and immunogenicity of rKVAC85B in a BCG prime-boost regimen against H37Rv and HN878 Mycobacterium tuberculosis strainsPLOS 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?

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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 describes a novel vaccinal construct for vaccination against Tuberculosis. It is a novel attenuated vaccinia virus (KVAC103) expressing Ag85B (rKVAC85B). They analyzed the immunogenicity and the efficacy of the vaccine, also in a prime-boost inoculation strategies with BCG in a murine model of TB. the work is quite interesting even if not very original, unfortunately it has serious experimental limitations.

Major points:

1- In all the figures 2, 3, 4 e 5 only data "specific" for Ag85B are reported. The baseline responses (e.g. only culture medium) are missing. These controls are required to evaluate the baseline activation of cells and the increse/decrease due to Ag85B restimulation.

2- In Figure 2, the data related to the vaccination with BCG+rKVAC85B alone are missing. Is the prime with BCG enhances, reduces or leaves inaltered the Ag85B responses induced by rKVAC85B alone?

3- In all the figures 3, 4 e 5 the data related to the control vaccination with rKVAC85B alone are missing. An experiment in which all the various vaccine combinations are present at the same time is necessary to be able to make adequate comparisons. The data are required to explain the data showed in Figure 6 and Figure 7 (MGIA and in vivo MTB infection). Is the prime with BCG enhances, reduces or leaves inaltered the Ag85B responses induced by rKVAC85B alone?

4- Spleen cells of BCG-vaccinated mice are not good responder to Ag85B protein; ex vivo Ag85B restimulation can even inhibit the basal cytokine production by cells of BCG-vaccinated mice. Therefore, knowing not only the basal level of cytokine production but also that induced by stimulation with other MTB antigens, such as PPD, is essential to understand how the vaccine rKVAC85B modulates the entire BCG-mediated immune response.

5- Evaluation of Ag85B-specific immune responses after 1 week from the last immunization is not idoneous to reveal memory immunity, which is a priority to demonstrate the vaccine efficacy. Longer times need to be studied.6- In MGIA experiments (Figure 6), as well as in in vivo experiments (Figure 7) the data related to vaccination with rKVAC85B alone are missing. Is vaccination with rKVAC85B alone protective?

7- In MGIA experiments (Figure 6), the “potential” presence of BCG in spleen cells of BCG- or BCG+rKVAC85B-vaccinated mice, prior to in vitro BCG infection, needs to be evaluated. Do the spleens of mice vaccinated with BCG or BCG+rKVAC85B harbor live BCG at the time of organ explantation?

8- In Figure 7, why the CFU are higher in the lungs of mice infected with H37Rv instead of those infected with the hypervirulent HN878 strain? Furthermore, histology data should be graphed and an appropriate statistical analysis performed to demonstrate that the inflammation and area occupied by granulomas are higher in BCG- than in BCG+rKVAC85B-vaccinated mice.

9-In the Discussion, lines 398-417 and 420-444 are appropriate for the Introduction section.

10-in the Discussion, it is reported that “the increase in the IFN-γ levels following rKVAC85B vaccination highlights the vaccine's potential to elicit a strong immune defense against intracellular infections”, lines 449-450. Although the protective role of IFN-γ in TB is undoubted, many papers indicate that this cytokines needs to be balanced and its enhancement does not always correlate with better protection against Mtb infection. It is also true for the IFN-γ generated in response to Ag85B protein. This point should be discussed with greater attention also considering that there is no data in the work which supports the relationship between the increase in IFN-γ and protection.

Minor points

1- Add refs in the introduction section line 82

Reviewer #2: Reviewer's Comments on Manuscript PONE-D-24-18487

Title: Efficacy and Immunogenicity of rKVAC85B in a BCG Prime-Boost Regimen Against H37Rv and HN878 Mycobacterium Tuberculosis Strains

General Assessment:

The manuscript presents a study on the efficacy and immunogenicity of a recombinant vaccinia virus expressing Ag85B of M. tuberculosis (rKVAC85B) employing two prime-boost regimens against two strains of M. tuberculosis: standard laboratory H37Rv and the hypervirulent HN878 strains. The research is timely and addresses a critical need for improved tuberculosis vaccines, especially considering the variable efficacy of BCG in adults.

Strengths:

The study introduces a novel vaccine candidate using KVAC103, an attenuated vaccinia virus as vaccine vector to express Ag85B, which is of significant interest in the field of TB vaccine research. The focus on both H37Rv and hypervirulent HN878 strains enhances the study's relevance. The study adheres to ethical standards, with appropriate approvals and descriptions of animal welfare considerations.

Areas for Improvement:

1. Introduction:

While the existing introduction provides a wide background but could benefit from more detailed but specific information on the limitations of BCG and other current TB vaccines in clinical trial, and the specific advantages of using vaccinia virus vectors and the specific antigen (Ag85B) and its specific role in M. tuberculosis.

2. Materials and Methods:

The M&M section requires further comprehensive details. For instance:

(i) the description of the generation of rKVAC85B could include more details on the verification steps (e.g., PCR conditions, primer sequences, and agarose gel images) as supporting data, such that it should be easy to follow the protocols by other researcher or reader.

(ii) the authors should provide clear pictorial (cartoon) outlines of the immunogenicity and protective efficacy study protocols as part of the respective result figures, for greater clarity to the reader.

(iii) the methodology for the bead-based cytokine analysis could be expanded to include the rationale for selecting these cytokines.

(iv) while MGIA is an interesting and powerful ex vivo assay to measure the efficacy of cellular immunity in controlling bacterial growth, inclusion of BCG as the target bacteria reduces its significance in this study that targets to evaluate the efficacy of new vaccine against two virulent strains of M. tuberculosis. This requires appropriate rationale or redoing it with virulent M. tuberculosis.

3. Results:

The description of results and the respective figures need appropriate revision. For instance:

(i) the immunogenicity experiment seems to be conducted for all five groups together (Unvaccinated, BCG, rKVAC85B, rKVAC85B prime boost, and BCG- KVAC85B prime boost) as described in the M&M section (Line 150). However, results were depicted separately which makes it difficult to compare among all the groups. Animal numbers in each group for immunogenicity study was not mentioned, while for efficacy study it is mentioned as (n=5). Figure 2 C and D, and Figure 4 A and B bar diagrams should be provided with the error bars and appropriate statistics to show significance of the observed differences. The conc. of Ag85B protein that was used as the coating/stimulating agent not mentioned. Details of Ag85B epitope/peptides used for ELISPOT assay should be provided. Figure 5, IL-10 levels were depicted, typically this is a Th2 cytokine and known to enhance pathogen favoured response in TB, however, its heightened induction in the case of BCG prime rKVAC85B was not explained well. Information on number of samples, technical replicates should be provided.

(ii) as pointed out above, use of BCG as the target pathogen in the MGIA requires justification.

(iii) typically, in the mouse model of TB vaccine efficacy studies, both lungs and spleen CFU counts are provided. In this study, only lung CFU was shown. Speen CFU data highlights how the vaccination would prevent dissemination of infecting pathogen to distant lymphoid organ. It would be better to show both lung and spleen CFU data. Further, in the absence of details of lungs homogenisation and plating protocol (which/how many lobes for what purpose, weight of lungs) it is difficult to appreciate the CFU data presentation as Log10 CFU/ml as weight/size of lungs varies between vaccinated and unvaccinated animals. Figure 7 needs major revision, it is appropriate to depict CFU data as per lungs or per gram of tissue, CFU data for all the groups in the study is warranted, labelling of the unvaccinated and infected group as normal is technically incorrect, should be revised.

4. Discussion:

The discussion could be strengthened by more critically comparing the results with existing studies on TB vaccines, particularly those using similar viral vectors, and explaining why the vaccine under study could be superior. Discuss the conundrum of heightened L-10 response in the study. Also, highlight future research directions.

Minor Comments:

(i) Ensure consistency in the use of abbreviations throughout the manuscript.

(ii) A few typographical and grammatical errors need correction for clarity and readability.

(iii) Ensure all references are up-to-date and formatted according to the journal's guidelines.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-18487R1Efficacy and immunogenicity of rKVAC85B in a BCG prime-boost regimen against H37Rv and HN878 Mycobacterium tuberculosis strainsPLOS ONE

Dear Dr. Jeong,

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 Feb 21 2025 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,

Syed Faisal, PhD

Academic Editor

PLOS ONE

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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.

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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: (No Response)

Reviewer #2: All comments have been addressed

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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 #2: Yes

**********

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

Reviewer #1: (No Response)

Reviewer #2: Yes

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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

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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

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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: Although some parts have been improved, the revised version of the manuscript did not address the various criticisms raised by the reviewers, especially those that would have required novel experiments. In particular, the authors did not provide comparable data across all the different groups investigated (PBS, rKVAC85B, rKVAC85B boosting, BCG and BCG-rKVAC85B) that would have allowed a complete and correct comparison to assert that boosting with rKVAC85B enhanced the vaccine efficacy of BCG. Also the lack of data on the basal levels of the various immunological parameters analyzed as well as the disparity of the vaccination protocols used do not allow to clarify what was actually modulated. Furthermore, the histology figures of the lungs of the infected mice do not seem very consistent with what has been reported in panel D of Figure 7.

Reviewer #2: The revised manuscript is substancially improved. Except some typographycal errors that requires rectification, there is no more comments.

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Reviewer #1: No

Reviewer #2: No

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Efficacy and immunogenicity of rKVAC85B in a BCG prime-boost regimen against H37Rv and HN878 Mycobacterium tuberculosis strains

PONE-D-24-18487R2

Dear Dr. Jeong,

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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Kind regards,

Syed Faisal, PhD

Academic Editor

PLOS ONE

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Reviewers' comments: