PONE-D-23-43523Relationship between protein conformational stability and its immunogenicity when administering antigens to mice using adjuvants - Analysis employed the CH2 domain in human antibodies -PLOS ONE

Dear Dr. Ueda,

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 Apr 19 2024 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,

Rui Tada, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (a) methods of sacrifice, (b) methods of anesthesia and/or analgesia, and (c) efforts to alleviate suffering.

3. Thank you for stating in your Funding Statement: 

This work was supported by the MEXT (Ministry of Education, Culture, Sports, Science, and Technology) Grant, Kyushu University operating expenses, and under the “COVID-19 Drug and Vaccine Development Donation Account” Project from Sumitomo Mitsui Trust Bank.

Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. 

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

This work was supported by the MEXT (Ministry of Education, Culture, Sports, Science, and Technology) Grant, Kyushu University operating expenses, and under the “COVID-19 Drug and Vaccine Development Donation Account” Project from Sumitomo Mitsui Trust Bank.  We would like to thank Dr. Takahashi and Prof. Caaveiro from Kyushu University for their kind guidance in the experimental procedure.

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

This work was supported by the MEXT (Ministry of Education, Culture, Sports, Science, and Technology) Grant, Kyushu University operating expenses, and under the “COVID-19 Drug and Vaccine Development Donation Account” Project from Sumitomo Mitsui Trust Bank.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript.

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

6. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

[Note: HTML markup is below. Please do not edit.]

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: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

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

**********

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

**********

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 work "Relationship between protein conformational stability and its immunogenicity when administering antigens to mice using adjuvants - Analysis employed the CH2 domain in human antibodies"carefully describes the importance of the stability of antigens for antibody production that vary depending on the use of different adjuvants .Another point to remember is that different proteins require stabilisation to activate the immune response. The role of the djuvant used is effective in stimulating mediated immunity both for the potentiation of T helper cells that lead to the production of different types of immunoglobulins and the effective participation of effector T cells.The authors also point out that it is important to note that the stability of the antigens used in an immunisation scheme will have to be standardised, the antigens for the production of antibodies and in the case of mice the importance of the strain chosen for the type of response expected. In previous studies "Zhang N, Channappanavar R, Ma C, Wang L, Tang J, Garron T, et al. evaluated adjuvants for receptor-binding domain-based subunit vaccines against Middle East respiratory syndrome virus". Cell Mol Immunol. 2016;13: 180-190. doi:10.1038/cmi.2015.03 thus showing the importance of the adjuvant in modulating the immune response.

The experimental design is adequate, but there is some error in the text.

Please review

line 118 PolyHYDROXYETHYL A column (200 Å,

line 126 using an AutoflexÅ mass spectrometer (Bruker Daltonics, Germany) w

Reviewer #2: General comment: The authors have made an attempt to elucidate the stability of proteins and their immunogenicity. Overall, the rationale and study design are quite interesting. However, further clarification and improvements in manuscript writing are needed.

Specific comments:

Abstract:

1. The abstract should be based solely on your findings, e.g., anti-drug antibodies are not directly related to this study.

2. The abstract should not include discussion points, such as “The findings, in conjunction with a previous study, indicate that protein stability plays a crucial role in eliciting an immune response in mice through the binding of protein antigens to B cell receptors on APCs.”

3. There is no supporting data from this study for “It is important to note that the optimal stability of antigens for antibody production is anticipated to vary depending on the use of different adjuvants in animal experiments.”

Introduction:

4. The mechanism of action of Alum and IFA/CFA is required, as they might have different immune stimulation pathways, which could lead to differences in immune induction capability (not solely due to the effect of protein stability).

5. RNase A and RNase S were not mentioned.

6. Information about mut20 (mutation position, changes in disulfide bonds, etc.) and its conformation (compared to the wild-type) must be included.

Materials and Methods:

7. Producing mut20 in yeast could potentially yield a glycosylated protein. How did the authors avoid the effect of the glycosylation status of these two proteins on antibody induction capability?

8. The statement “All other chemicals used in the study were of the highest commercial quality available” is vague. How can the authors ensure they were the best quality on the market?

9. The immunization schedule was quite short (1-week intervals). For protein (and other platform) vaccine immunization, at least a 2-week interval is usually employed to allow immune cell maturation.

10. The approved animal protocol number should be included.

11. Why was the intraperitoneal route used instead of the intramuscular route? This does not reflect the standard immunization procedure.

12. Why were different protein amounts used for adjuvanted and non-adjuvanted groups (30 vs. 100 micrograms)? We cannot directly compare the magnitude of the antibody response as the vaccine doses differ.

13. What is the reason for using CFA in the first immunization and IFA for the booster? Please explain and state this in the manuscript.

14. Salt concentrations in buffers must be indicated as their final concentrations (mol/liter, molar), not in grams.

15. Line 105, what is the initial dilution, and how did you dilute the tested serum?

16. ELISA: The methods for stopping the reaction and reporting readouts are missing. Was the background subtracted?

Results:

17. For all results sections, please do not repeat the introduction and method. Authors must directly explain the obtained results concisely (important). Also, the discussion should be stated separately in the discussion section.

18. For better understanding, CH2, mut20, RNase A, and RNase S should be indicated in Figures 3C-3F.

19. Again, authors never mentioned and explained about RNase A/RNase S; it suddenly appeared in lines 165-177.

20. Where is the data to support the statement? “When examining the MHC-peptide complex in APCs and T cell presentation, it is suggested that the immunogenicity of RNase A compared to RNase S is not solely determined by the degree of protease digestion of the RNases.”

Discussion:

21. More discussion is needed on topics other than protein stability, including adjuvant properties. Moreover, as the authors tested in BALB/c mice, which is an inbred strain with a restricted TCR and MHC repertoire, further investigation in an outbred mouse strain may be needed to confirm your findings.

22. What is the explanation for the opposite IgG results when the adjuvant is changed (Figure 1B)?

**********

6. 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: Yes: Eakachai Prompetchara

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Response to authors-PONE-D-23-43523 .docx

PONE-D-23-43523R1Relationship between protein conformational stability and its immunogenicity when administering antigens to mice using adjuvants - Analysis employed the CH2 domain in human antibodies -PLOS ONE

Dear Dr. Ueda,

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.

==============================

Thank you for submitting your revised manuscript. We appreciate the effort you have put into addressing the comments from Reviewer 2.

Unfortunately, despite multiple invitations and reminders, we did not receive a response from Reviewer 1 during this round of revisions. However, upon careful examination of Reviewer 1's initial comments, we found them to be minor in nature and have determined that you have already adequately addressed them in your revised manuscript.

Considering the thoroughness of your revisions in response to Reviewer 2's comments and the minor nature of Reviewer 1's suggestions, we believe that your manuscript is now suitable for publication after addressing the remaining minor points raised by Reviewer 2.

Please submit your revised manuscript, along with a detailed response letter addressing the comments from Reviewer 2. We look forward to receiving your revised manuscript.

==============================

Please submit your revised manuscript by Jul 06 2024 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,

Rui Tada, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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.

[Note: HTML markup is below. Please do not edit.]

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 #2: (No Response)

**********

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 #2: Yes

**********

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

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 #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 #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 #2: The manuscript has improved, yet there are a few areas that require further clarification to enhance the understanding of the experiments conducted and the findings reported:

Comment 7: Please include the results pertaining to the isolation of non-glycosylated proteins. It would be appropriate to add these details in the supplementary data section. Additionally, clarify how you confirmed the identity of the expressed protein as CH2. Were Western blotting techniques used for this purpose? Please elaborate.

Comments 9 and 11: It is necessary to add the reference for the animal protocol that was previously referred to by the authors. This will help in maintaining the integrity of the experimental framework.

Comments 12 and 13: I recommend that you integrate a more detailed explanation regarding these specific concerns within the discussion section of the manuscript. Please ensure that relevant references are cited to support your explanations.

Comment 16: This detail should be included in the methods section of your paper to provide clarity on the procedures used in your study.

Comment 21: The discussion concerning the choice of mouse strain should remain within the discussion section of the manuscript. This will help readers understand the rationale behind the selection and its relevance to the study outcomes.

**********

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 #2: Yes: Eakachai Prompetchara

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Relationship between protein conformational stability and its immunogenicity when administering antigens to mice using adjuvants - Analysis employed the CH2 domain in human antibodies -

PONE-D-23-43523R2

Dear Dr. Ueda,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Rui Tada, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: