PNTD-D-24-01702

A composite subunit vaccine confers full protection against Buruli ulcer disease in the mouse footpad model of Mycobacterium ulcerans infection

PLOS Neglected Tropical Diseases

Dear Dr. Reljic,

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We look forward to receiving your revised manuscript.

Kind regards,

Elsio A Wunder Jr, DVM, Ph.D.

Section Editor

PLOS Neglected Tropical Diseases

Elsio Wunder Jr

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

Additional Editor Comments (if provided):

Journal Requirements:

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Reviewers' Comments:

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Objectives are clear and inline with the hypothesis stated. The study design is appropriate to address the objectives. To enhance reproducibility, provide more detail on the specific assays used to measure immune response.

There are no ethical concerns.

Reviewer #2: (No Response)

Reviewer #3: The objectives of the study was well articulate and thoroughly described.

The study is appropriate for the set objectives. The number of mice in each group of immunisation arm is not clearly indicated but can be only inferred from Fig. 5 as 6 or 5 mice per arm. This has to be clearly indicated in the method section.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The analyses presented are robust.

Reviewer #2: (No Response)

Reviewer #3: The results are clearly and appropriate presented with figures and images.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: All conclusions are supported by the results.

Reviewer #2: (No Response)

Reviewer #3: The conclusions are supported by the data presented and study limitations are also described. Overall the study set the stage for further investigation into the vaccine candidate for Buruli ulcer.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The very well written manuscript presents a significant contribution in the field of vaccine development against BU as the results are promising. The use of the composite sub-unit formulation is innovatives and relevant in the fight against BU.

The study design and use of the mouth foodpad model, is apropriate and results are compelling, demonstrating absolute protection in the treated groups.

The authors could address the potential challenges in translating these findings to human applications.

Reviewer #2: The manuscript by Boakye-Appiah et al. presents the use of a composite subunit vaccine formulation, named "Burulivac", in the mouse footpad model of Mycobacterium ulcerans infection. While the development of a vaccine for Buruli ulcer (BU) is an important area of neglected diseases research and the observed prevention of footpad ulceration in mice challenged with M. ulcerans after receiving Burulivac is interesting, the proposed vaccine strategy and evaluation of data have several significant shortcomings:

1. In the presented vaccine formulation, mycolactone, which is responsible for much of the pathology of BU, is included in its native, toxic form. What is the reasoning for using the native toxin rather than a modified, attenuated version? Several effective toxoid vaccines, such as those for diphtheria, tetanus, or botulism rely on inactivated forms of the toxin. To develop a vaccine that is safe for humans, stimulating an immune response without causing harm, it is essential to use an attenuated form of the toxin. Using the native, toxic form therefore does not appear to be a viable approach for developing a human vaccine for Buruli ulcer. Therefore, I do not agree with the authors’ main conclusion that Burulivac is a promising vaccine candidate for BU.

2. The authors state that they included mycolactone as the immune modulator but do not provide evidence for this role of mycolactone in their manuscript. While they assess immune responses in vaccinated mice against the two included protein antigens, they do not analyze responses against mycolactone. They mention that they did not directly test for any potential immune responses specific to mycolactone, stating that “it is a non-proteinaceous molecule and there are currently no established protocols to detect them”. However, other research groups have reported the development and use of ELISAs, which can determine antibody responses to mycolactone. Analyzing antibody responses to mycolactone after vaccination could provide valuable insights for improving future vaccine design. Considering that antibodies are crucial for anti-toxin immunity, antibody responses to mycolactone may be the defining factor in the observed protection. If this is not the case, it would be crucially important to characterize the proposed immune modulatory activity of mycolactone, as other groups have reported that strong immune responses against a range of M. ulcerans proteins confer no or only limited protective efficacy in the mouse footpad model.

3. The authors do not provide a clear rationale for their choice of the composite subunit vaccine formulation. It would be helpful if they could explain why they selected and combined Ag85A, KRA, and mycolactone. Furthermore, no dose-finding experiments have been reported to determine the optimal concentrations of these components. This is particularly important for mycolactone, to provide insight into its contribution to vaccine efficacy.

4. What is the rationale behind the choice of the different immunization regimens used in this study? Why were mice in Arm A groups 1 to 3 immunized only once with BCG and Mu delta, while groups 4 and 5 were immunized 3 times (according to Table 1)? This discrepancy makes a valid comparison of the performance of vaccine formulations to prevent footpad ulceration across the groups in Arm A impossible. It is well-established that boosting leads to more effective immune responses, so this variation in regimen complicates the interpretation of results. Essentially, there is no valid comparative regimen to the proposed Burulivac regimen. Group 4 was first immunized with Mu delta and then boosted with Burulivac so even Mu delta vaccination cannot be directly compared to Burulivac. Therefore the performance of Burulivac compared to all other regimens remains unclear.

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes:  Michael Frimpong

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Dear Dr. Reljic,

We are pleased to inform you that your manuscript 'A composite subunit vaccine confers full protection against Buruli ulcer disease in the mouse footpad model of Mycobacterium ulcerans infection' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Elsio A Wunder Jr, DVM, Ph.D.

Section Editor

PLOS Neglected Tropical Diseases

Elsio Wunder Jr

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #3: The authors have addressed all comments raised previously by reviewer and updated the manuscript accordingly.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #3: (No Response)

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #3: (No Response)

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #3: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #3: (No Response)

**********

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 #3: Yes:  Dr. Michael Frimpong