PONE-D-20-33627

Physical Properties of Lactic Acid Bacteria Influence the Level of Protection Against Influenza Infection in Mice

PLOS ONE

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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: 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?

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: 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 paper is of some interest and provides evidence that more effective dispersion of a killed lactic acid bacteria preparation is possible and that this leads to greater activity in an in vivo model vs influenza virus. The main positive findings include reduced viral load in BALF samples at day 3 (and to a lesser extent in whole lung samples) and increased antiviral IgA response compared with non dispersed bacteria. There is evidence of increased IL-2 production with the dispersed preparation from isolated spleen cells. The topic is of interest especially since this seems to be a potentially benign way of reducing severity of lung viral infection through oral ingestion of killed and powderized bacteria. The critiques are as follows:

1. The microscopy seems to be of poor quality and it is hard to assess in the images the location of the intestinal cells in the background. The confocal microscopy is somewhat better but it is necessary to show the results of the aggregated bacteria on confocal microscopy as well as the dispersed.

2. It should be elucidated what the basis for assuming that the bacterial mechanism of action mainly involved binding to Peyer's Patches.

3. It should be made clear in the discussion that the theory that the aggregated bacteria cannot traverse mucin layers in speculative.

4. The discussion also notes that there is different interaction of the dispersed bacteria with macrophages and dendritic cells but it is not clear from the data presented that these are the cells involved in generating the IL-12.

5. There should be some discussion of why the splenocyte and IL-12 experiments were done.

6. It should be made clear if the rate of recovery of weight was statistically different for the dispersed bacteria.

7. Figure 4 title refers to ribonuclease treatment - Where does the ribonuclease come in?

Reviewer #2: In this study, the authors investigated how the powderization of lactic acid bacteria (LAB) affects their water dispersibility and their efficacy in inducing immune response in a mouse model of IFV infection. Two different types of powdered Enterococcus faecalis KH2 preparation (non-treated and dispersed) were compared in their particle size, the distribution in the small intestine and the protective effects of these LAB preparations against viral infection in a mouse model of influenza infection. The authors showed that by treating LAB with a high-pressure homogenizer and adding dextrin in the preparation can dramatically decrease the formation of bacteria aggregates, which increased the water dispersibility of the bacteria and thus enhanced their uptake by intestinal Peyer’s patches and improved their protection against virus infection. The experiments were well-designed, and the conclusion was reasonably sound. However, there are several major concerns that should be addressed:

1. Several abbreviations were not defined in the manuscript and should be added in the main text where it first appeared (eg. PRRs, M cells, et al).

2. Line 62, “to investigate whether the effect of the difference in the LAB species or powderization was due to the difference in the species” This statement is really confusing and should be rephrased. Also, were the species of bacteria in the LAB powdered preparation different in n-LAB and d-LAB?

3. Typos and grammar issues should be double-checked and corrected.

4. In the evaluation of LAB’s distribution in mouse Peyer’s patches, male specific pathogen-free mice were used, whereas female mice were used in the influenza infection model. Are there any particular reasons in choosing female mice in the infection model? And have you looked at the bacteria distribution in Peyer’s patches in female mice as well? Is it different from male mice, can you comment on this?

5. The distribution of LAB in mouse intestine was evaluated in male mice injected with LAB and harvested after 1h in this manuscript, LAB preparations were also administered to female mice by gavaging in the IFV infection model. Have you looked at the distribution of LAB in mice with IFV infection? How does the distribution change overtime before and after virus infection? Can you comment on this?

6. Line 99, the LAB suspension was added “to six wells of a 96-well cell culture plate”, is this correct?

7. In the IFV infection model, n-LAB, d-LAB and OSL were given two times per day from 7 days before viral inoculation to 14 days after inoculation. Were there any differences in body weight change in the first 7 days with LAB administration before viral inoculation? Have you tried different treatment schemes (pre-treatment, LAB administration during infection, or LAB after infection) with LAB preparations in the infection model and were there any differences?

8. Figure legends should be placed following the figure or combined and placed after the main text of the manuscript.

9. Virus loads in BALFs and lungs were determined on day 3 after IFV infection and neutralizing antibody and IgA levels were determined on day 14. Have you tested the virus load in BALFs and lungs on day 3 as well?

10. In Figure 3, a and b seems showing same magnification based on the scale bar, however the images seem to be taken under obviously different magnification scale, this should be addressed and corrected, if any applicable. Additionally, the indications of different fluorescence colors presenting in Figure 3c should be added in the figure legend.

11. Line 181-184. This part of result was written poorly. The rationale and hypothesis of this specific experiments set should be addressed and a conclusion from the observations should be made or at least discussed.

12. Influenza infected mice treated with d-LAB lost weight at a slower rate and recovered faster compared to those treated with n-LAB, was the difference statistically significant? The signifiers should be added to the graphs if there is any.

13. Figure 6a, significance bar misplaced. Please correct this.

14. Line 207, The d-LAB group had a significantly lower viral load than n-LAB group in the BALFs but no differences were seen in the lung virus yields, can you explain this?

15. A control group of influenza infected mice treated with LAB culture preparation without powderization should be added, in comparison to different types of powdered LAB preparations.

16. Are there any other aspects other than the formation of aggregates that could possibly affect the beneficial effects of LAB? (eg, bacteria morphology changes during and after powderization, biofilm formation, et al.)

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Reviewer #1: Yes: Kevan Hartshorn

Reviewer #2: No

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Physical properties of lactic acid bacteria influence the level of protection against influenza infection in mice

PONE-D-20-33627R1

Dear Dr. Watanabe

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.

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Kind regards,

Wenke Feng, 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: (No Response)

Reviewer #2: The authors have addressed all the comments and questions in a comprehensive way, the suggested information were also added to the main text.

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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: Yes: Kevan Hartshorn

Reviewer #2: No