PONE-D-23-39898Salivary immune responses after COVID-19 vaccinationPLOS ONE

Dear Dr. Vinjé,

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. Although the topic is interesting, but One reviewer has raised serious concerns.

Please submit your revised manuscript by Mar 07 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Gheyath K. Nasrallah

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: Partly

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: 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: This is an interesting study whose objective was to evaluate the salivary IgA and IgG immune response after vaccination with the COVID-19 Moderna mRNA-1273 vaccine. Their results confirm the potential utility of saliva in assessing immune responses after immunization and highlights that testing could lead to novel insight on mucosal immune response against SARS-CoV-2.

MINOR REVISION:

The infection status of the individuals before or after vaccination is not mentioned along the writing. Figure 4 is showing reactivity to nucleocapsid for some individuals and in Figure 3, saliva samples showed positivity at day 0 and 7, indicating that some patients had viral exposure within or before the study period.

Because SARS-CoV-2 infections occurring before, between or after doses could influence the level and persistence of anti-SARS-CoV-2 IgA or IgG antibodies in saliva and blood specimens, it would be advisable to incorporate this information into the study.

Additionally, it would be important to conduct analyses comparing individuals who experienced infections with those who did not, to recognize if there are any significant differences. Or at least justify why it is not shown.

Reviewer #2: Dear authors, please address the following points:

- line 111 authors states they used pre-pandemic saliva samples as negative control, please clarify the source of these sample, where they came from a bank of saliva sample collected from generic patients before pandemic? How were they stored?

-line 125 authors wrote:"participants self-collected whole blood", in my opinion it is worthy specify that the collected sample are Capillary blood sample;

- line 132 remove one square bracket;

- line 174 the total number of 1023 seems wrong. (200-42)*6=948. If you excluded 42 patients from the original 200, it means you analyzed 158 patients. So the total number of collected samples should be 948.

- line 178. In table 1 you should report information on the 158 patients really enrolled in the study excluding information of patients excluded.

- line 186 The dot at the end of the line is a typo. A dot instead should be inserted at the end of line 187.

- line 191 Authors stated they collected blood samples from 127 partecipants, was this blood collected from a venipuncture procedure? Please clarify.

- line 194 and line 254. Authors analyzed data reporting median and percentile, meaning data are not distributed normally. But they performed ANOVA test, that is usually used for data distributed normally. Can authors please clarify and justify the statistical test performed?

Reviewer #3: The manuscript reports induction of salivary antibody titers upon vaccination against SARS-CoV-2. Authors report significant increase in anti-Spike IgA and IgG two weeks after vaccination. It is a bit surprising, given that the development of antibody response takes around 2-3 weeks. This suggests that antibody response detected in these patients is driven by memory B cells, but not by naive B cells. The data on the status of participants are required: were these donors previously infected? Figure 4 reports some of the data that can be used rto determine the status, but these data are measured at day 42 after vaccination. It is of particulary importance for the data interpetation, since we have recently shown that in non-exposed participants salivary immune responses develop mcuh later (Bondareva et al., Cell Host and Microbe, 2023). Also, authors do not provide the levels of toal IgA/IgG in saliva. These parameters may vary quite extensively in saliva during collection and the normalisation to total IgA/IgG levels may further show whether this effect due to increase of specific antibodies or total antibody levels (bondrave et al., J of autoimmunty, 2022).

Minor comments:

1. Authors describe in the introduction the salivary Ab measurement as easier way to estimate blood Ig levels, but do not really mention that Ab in the oral cavity are of physiological significance, since they do actually protect against the initial virus infection. I would add this in introduction with the relevant quotes.

2. Authors do not cite a lot of relevant literature that was focused on salivary Ab responses, including series of works from J. Gommermann, and others. Thus, discussion should be extended and should include all the relevant papers published on this topic. So far it is quite poor and ignores many seminal works addressing the kinteic and importance of these responses.

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Reviewer #1: No

Reviewer #2: Yes: Eleonora Nicolai

Reviewer #3: Yes: andrey kruglov

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PONE-D-23-39898R1Salivary immune responses after COVID-19 vaccinationPLOS ONE

Dear Dr. Vinjé,

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.

While we appreciate the effort you put into revising your manuscript based on the reviewers' feedback, I regret to inform you that we are unable to accept the manuscript for publication at this time. One of the major comments raised by one of the reviewers has not been adequately addressed in the revised version. I cannot proceed with the publication until all major concerns raised by the reviewers have been satisfactorily resolved.

Please submit your revised manuscript by Jun 14 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,

Gheyath K. Nasrallah

Academic Editor

PLOS ONE

Additional Editor Comments:

While we appreciate the effort you put into revising your manuscript based on the reviewers' feedback, I regret to inform you that we are unable to accept the manuscript for publication at this time. One of the major comments raised by the reviewers has not been adequately addressed in the revised version. I cannot proceed with the publication until all major concerns raised by the reviewers have been satisfactorily resolved.

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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: All comments have been addressed

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: No

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

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: Yes

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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

Reviewer #3: 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: Dear Editor, in my opinion the authors have addressed all the questions raised and now the paper is suitable for publication.

Best regards

Reviewer #3: As it became evident during revision, the previous exposure to SARS-CoV-2 by participants cannot be ruled out. Thus, the conclusions about the kinetics of immune response upon vaccination cannot be made. In my opinion, this is a crucial point of the paper. Authors could have critically evaluated their data based on the already published reports, where clinical status was well documented. However, they also did not provide proper discussion in the revised manuscript and do not cite relevant literature.

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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 #2: Yes: Eleonora Nicolai

Reviewer #3: No

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[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.

Salivary immune responses after COVID-19 vaccination

PONE-D-23-39898R2

Dear Dr. Vinie,

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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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,

Gheyath K. Nasrallah

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 #3: 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 #3: Yes

**********

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

Reviewer #3: 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 #3: (No Response)

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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 #3: 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 #3: All the comments have been addressed. The manuscript can be accepted for the publication in Plos One.

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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 #3: Yes: Andrey Kruglov

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