PONE-D-21-11729

Effectiveness and Safety of oral lactococci-based vaccine encoding triple common allergens to prevent airway allergy in mice

PLOS ONE

Dear Dr. Chen,

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Regarding the experiments, the authors should complete the data by including the evaluation of antibodies (IgE, IgA and IgG2a) in samples collected from the airways (lung supernatants or BAL, for instance).

The authors should also comment on the interval chosen for analyzes of the adverse effects measured by histological evaluation of organs as well as other parameters that could be applied.

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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?

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: Yes

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

Reviewer #1: N/A

Reviewer #2: Yes

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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

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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: No

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: I find the manuscript interesting, considering the approach of a potential preventive strategy to avoid allergies to certain allergens. The studies carried out both to obtain recombinant L. lactis for 3 antigens and the assays in a mice experimental model involve laborious work. However, I have some suggestions and inquiries to the authors:

1- I suggest to improve the introduction, to avoid the repetition of concepts (lines from 50 to 59)

2- L. lactis is simple to cultivate and it has the ability to secrete recombinant proteins to the growth medium, which together facilitates downstream processing of recombinant protein. In this work the proteins are not secreted, are produced in cytoplasm. If this is so, it should be clearly redacted in Material and Methods.

3- The authors evaluated the potential adverse effects of vaccination outside the lung through very general parameters such as weight of mice, weight of spleen, liver and kidney. It is likely that the animals will need to be monitored longer to see changes in these parameters. Histological studies were carried out in kidney and liver and they are important. However, there are simple serological parameters that provide specific data on kidney (creatinine levels in serum) and liver functionality (AST and ALT enzyme blood levels), for example. It would have been interesting to evaluate these parameters.

4- The IgE and IgG2a levels were evaluated in serum. I consider it is important to evaluate these parameters in bronchoalveolar lavage (BAL).

5- Methacholine is an inhaled drug that causes a slight narrowing of the airways in the lungs. The methacholine challenge test is a type of bronchial challenge test used to help diagnose asthma. I think that to include a brief sentence about this would facilitate the understanding of non-expert researchers on the subject.

5- In Table 2: Il should be changed by IL.

6- In Figures 4 (AB, CD, EF) Is it possible used the same scale for IgE and IgG2a?

7- The authors mentioned the limitations of this study but considering globally the results, I think the discussion could be improved.

Reviewer #2: The manuscript is well-written and describes, in a concise way, the aims of this investigation. Vaccine-based interventions represent a cost-effective alternative to prevent and treat many diseases, especially those related to hypersensitivities. Although this formulation is not the pioneer in the prevention or treatment of allergies, the research's originality relies on a combined allergens (expressed in GRAS) strategy. In this context, the work is relevant and consists of an original contribution to a preventive as well as potential therapeutic use of vaccines. The manuscript also reveals a substantial amount of work and the use of adequate techniques to characterize their system.

There are just some minor questions that would be addressed by the authors:

1. In Materials and Methods (lines 169-180), the authors describe the immunization protocol, with specific doses, besides the sensitization and challenge methods. It is not clear how they made this experimental design, since this section does not bring any references. These protocols, mentioned previously, were not referenced. Therefore, I recommend that the authors add more information about the experimental design regarding these methods.

2. The information of protein sizes (in kDa) is lacking in the text (Materials and Methods and Results as well). I suggest that this piece of information appears in Results section (for instance, between lines 246-254).

3. The statistical analysis was not properly described in the text (lines 240-243). The authors only mention that "appropriate methods" were performed to analyze the results. The specific information about statistics of antibodies levels can be found only in the figure 4 description, for instance.

4. I was wondering why the authors did not collect nasal as well as bronchoalveolar lavages to determine IgA levels. Hypersensitivities that affect respiratory tracts (upper and lower) usually result in local and systemic response, characterized by IgG (from Th2 profile), IgE and secretory IgA production. For this reason, the authors would address this question, in other words, they should clarify the reason why this isotype was not analyzed.

If the authors address these questions, the manuscript will be suitable for publication at PLoS One.

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Reviewer #1: No

Reviewer #2: No

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Effectiveness and Safety of oral lactococci-based vaccine encoding triple common allergens to prevent airway allergy in mice

PONE-D-21-11729R1

Dear Dr. Chen,

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,

Maria Leonor S Oliveira, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: 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 #2: 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 #2: Yes

**********

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: The manuscript was substantially improved.

I think that the authors responded favorably to the suggestions and questions raised, and in its current form, the manuscript should be accepted for publication.

Reviewer #2: The authors presented a document with all comments addressed. I would like to add some minor correction regarding the answer to question 4 of my comments. It is a matter of misspelling. They must write "secretory" instead of "secretary" and "requires" instead of "required". In spite of this minor issue, the paper is ready for publication.

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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 #2: No