PONE-D-22-11120Food and feed safety of the Bacillus thuringiensis derived protein Vpb4Da2, a novel protein for control of Western Corn RootwormPLOS ONE

Dear Dr. Wang,

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.

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Academic Editor Comments: Firstly, improve upon the English language of the MS starting from the abstract. Secondly, rectify the errors when citing the figures or the lanes in some of the gel (this is a serious issue as indicated by the reviewer 2). Thirdly, provide some background knowledge about the mode of action of VpbDa2 protein (how the protein is activated in the gut of target insect) in the introduction.    

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PLOS ONE

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Additional Editor Comments (if provided):

Firstly, improve upon the English language of the MS starting from the abstract. Secondly, rectify the errors when citing the figures or the lanes in some of the gel (this is a serious issue as indicated by the reviewer 2). Thirdly, provide some background knowledge about the mode of action of VpbDa2 protein (how the protein is activated in the gut of target insect) in the introduction.

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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: Yes

Reviewer #2: Yes

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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: In this article titled “Food and feed safety of the Bacillus thuringiensis derived protein Vpb4Da2, a novel protein for control of Western Corn Rootworm” the authors investigated the safety of a novel insecticidal protein, Vpb4Da2 derived from Bacillus thuringiensis. Overall, this article is well composed and could be published after few modifications,

1. It will be nice to mention about the requirements for passing a protein as safe for human consumption. What are the criterion set be regulatory bodies?

2. What is the mode of action of VIPs? It would be helpful to demonstrate the similarity and differences between the structure of VIP and CRYs

3. How much maize and E.coli was used to purify the proteins?

4. What was the buffer composition used to extract Vpb4Da2 from meize?

5. Centrifugation or filtration, which method was used to clarify the maize extract

6. What was the salt gradient?

7. How buffer exchange was done?

8. I am guessing it is anti- Vpb4Da2 mAB. From where did the authors get this?

9. L104, A little bit of detail about the cloning is needed. How the coding sequence was amplified, PCR conditions, restriction enzymes etc need to be mentioned,

10. L107, what was the composition of the neutral buffer?

11. L107, what kind of cell disruptor was used? How did you get the soluble fractions?

12. L109 Typo Ni-NTA, Source of the protease?

13. Source pepsin?

14. EC50 of E. coli expressed protein was

15. What is in the parenthesis in the EC50 values in table 3

16. L315, fig 5 was referred wrong as fig 3, same in 335. But there is no fog 5B either.

Reviewer #2: The authors study whether the VpbDa2 protein which has been described to be toxic to Western corn rootworm (WCR) can be safely used in agriculture. Indeed, some other insecticidal proteins derived from Bt are commonly used to control insect pests (either as Bt biopesticide formulations or as GM crops) and passed regulatory safety assessments. In this manuscript, the authors study if the Vpb4Da2 protein sequence has any similarity against known allergens and test the acute oral toxicity in mice. Also, they assess the Vpb4Da2 protein stability to pepsin and pancreatin (two different gut proteases) and to different temperatures.

Comments:

- The first section of the results “Characterization and equivalency assessments of Vpb4Da2 proteins” can be included in the material and methods section as they are comparing the protein coming from different sources (the GMO or the recombinant E. coli strain). If the authors do not want to move at least I will suggest moving some figures and Tables to supplementary material.

- Section “Heat treatment of Vpb4Da2 at temperatures common to cooking and processing”. The authors stated that “Vpb4Da2 retained partial insecticidal activity after incubation at temperatures up to 37 °C” (see lines 286-287). However, Table 3 shows similar values (almost identical) for the proteins incubated at 0ºC, 25, or 37ºC. Please modify the sentence. Then, the authors stated: “Additional bands at both higher and lower apparent MWs began to appear in samples heated at 55°C and became more prominent in samples heated at 75°C for both 15 and 30 minutes”. However, from the point of view of this reviewer, it is hard to see these bands in the gel provided. I would suggest making softer this affirmation to be more conservative. Please, try to adjust the lane numbers in figure 4. Also, why an arrow is pointing out the band in lane 7? From my point of view, you should point the main bands in lanes 7 and 8 (both lanes contain the control protein, isn’t it?

- Section “Susceptibility of the Vpb4Da2 protein to pepsin and pancreatin”. Please carefully revised the figures that you are citing, I think you should refer to figure 5 and not to figure 3. In a similar way, please check the lanes that you want to comment on in the text because they do not match.

Why did the authors use two different methods (SDS-Page and Western Blot) to check if the protein is degraded by the two different proteases? Please comment on that.

Most of the Bt proteins need to be activated by midgut proteases to exert their toxic action on the midgut of the target insects, do you know if the Vpb4Da2 protein needs to be activated by proteases? Has this protein a resistant core to midgut proteases like Cry1 proteins? This information would be interesting either in the introduction or in the discussion.

I do not have much experience in those experiments (protein stability to pepsin and pancreatin) but is any control normally used?

- Section “Acute toxicity testing of the Vpb4Da2 protein”. I will suggest moving the sentence “These results are consistent with those obtained from previous studies on 3-domain Cry proteins, β-pore-forming proteins, and Vip proteins conducted at high dose levels, respectively (Table 7) (Lanes 349 and 350) to the discussion section, as they are not part of the results.

- As the authors organized the discussions in sections, maybe it would be possible to mix the results with the discussion to make it clearer for readers.

Minor comments:

-Line 54, remove one extra parenthesis

- Please change Vip proteins for Vip3 proteins in the text. (i.e. lines: 70, 77…)

-Line 83, correct Cry3Bb1

Lanes 86-88, please add suitable references.

Lane 105, Changer “pET SUMO-his” to pET SUMO-His

Lane 107, Please described the neutral buffer (indicate the composition)

Lane 109, Please correct the name of the column (Ni-NTA column instead No-NTA column)

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Reviewer #1: No

Reviewer #2: No

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Food and feed safety of the Bacillus thuringiensis derived protein Vpb4Da2, a novel protein for control of Western Corn Rootworm

PONE-D-22-11120R1

Dear Dr. Wang,

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,

Tushar Kanti Dutta, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Kindly italicize Diabrotica virgifera virgifera throughout the manuscript during proof stage of the article.

Reviewers' comments: