PONE-D-20-16731

Avian antibodies (IgY) targeting spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibit receptor binding and viral replication

PLOS ONE

Dear Dr. Goepp,

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Paulo Lee Ho, Ph.D.

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: Partly

Reviewer #2: Yes

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

Reviewer #1: No

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: No

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: Reviewer Comments: The following corrections have to be done by the authors for the manuscript approval.

1. Line no.94 – mcg unit has to be corrected as “µg”

2. Line no.101: vaccine preparation – what is the adjuvant used for immunization during sensitization dose and Line no.104: booster immunizations - for booster dose on 14th day of immunizations?

- Clearly mention the antigen-adjuvant ratio preparations for immunization (v/v).

3. Line no.102: each breast of each of one hen pairing – Route of immunization? Intramuscular or subcutaneous has to be mentioned.

4. Line no. 105, 106: Protocols for hen maintenance, immunization, and phlebotomy were approved by the Scaled Microbiomics, LLC Animal Use and Care Committee – Ethical approval reference number need to be mentioned.

5. line no.107: Serum IgY antiviral reactivity:

Serum antibody titre representation using simple bar graphs with clear explanations to show the significant increase in antibody titre levels has to be shown.

6. Line no.124: Total protein concentration of egg yolk and serum:

Standard curve and dynamics of total protein concentration from both egg yolk and serum has to be represented graphically. Even simple graphs are sufficient but it should be clearly self explanatory as per the results obtained.

7. Line no.195: Avian IgY was successfully isolated from yolks, yielding up to 16.8l mg/ml

At which titre day egg this antibody yield was calculated?

8. Line no.196: from serum, yielding up to 23.1mg/ml

Similarly at which titre day of the serum collected post immunization, this antibody yield was calculated?

9. And from both the Line no.195, 196; are antigen-specific IgY antibodies titre has raised as per represented within a couple of week and only with one booster immunization? - JUSTIFY

10. Line no. 177: Plaque reduction assay – Show the results of cell lines and viral plaque titration evaluations.

11. Line no. 211: IgY titers in serum, with titers of 1:10,000 at weeks 3 and 4 (data not shown). – The data has to be represented in graphs showing the dynamics of significant increase in the antibody titration both in serum and the yolk.

12. Line no.166: Binding Inhibition of S1 to ACE2 and Line no. 216: Inhibition of S1 spike binding to ACE2 protein by S1-specific IgY – Show the resulting proof for inhibition. And Western blotting results are must to prove the binding efficacy and specificity of antibodies but it has not been submitted here. Show western blotting results also.

13. Figure 2: Titre of antibodies increased in serum and yolk at the same time period.? How is that possible? Because, it takes a week time for transfer of antibodies from serum to yolk. – Justify with proper results.

Reviewer #2: This manuscript presents excellent evidence for moving forward with an avian antibody approach to the treatment and/or prevention of COVID19. The authors demonstrated a robust and specific IG-Y immune response against SARS-CoV2 S1 spike protein. The approach described in the manuscript is well-conceived and the evidence is compelling. The data presented therefore support the author's conclusions. The statistical analysis is a standard one and it is appropriate for this experimental approach. The underlying PAGE gel data for the statistical analysis is a part of the manuscript.

I do think that discussion should be tweaked a bit: this evidence for this approach as compelling for the treatment of COVID19 based on the use of polyclonal antibodies in other clinical contexts is not germane to a PLOS ONE manuscript. So the discussion should be less speculative.

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Reviewer #1: No

Reviewer #2: No

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Avian antibodies (IgY) targeting spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibit receptor binding and viral replication

PONE-D-20-16731R1

Dear Dr. Goepp,

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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,

Paulo Lee Ho, Ph.D.

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 #2: All comments have been addressed

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 #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: N/A

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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: The authors have satisfied my concerns and I believe that this manuscript is now ready for publication. The design and execution of the experiments was well conceived. The results are interesting and will help advance our fight against the current pandemic.

Reviewer #3: The authors of this study have evaluated IgY antibodies as potential tool for passive immunotherapy against SARS-COV-2 through blocking the interaction between the virus and ACE2 receptor. There is one concern:

The main point in the manuscript is inhibition of adhesion of viral spike glycoproteins to human ACE2 protein receptors, so, evaluation of binding Inhibition of S1 to ACE2 in methodology section need further details

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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: No

Reviewer #3: No