Dear Prof. Ramezani,

Thank you very much for submitting your manuscript "Immunogenicity and safety of heterologous boost immunization with PastoCovac Plus® against COVID-19 in ChAdOx1-S or BBIBP-CorV primed individuals" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

The authors believed there is some value in comparing between different vaccine platforms. However, they raised several major concerns. In particular, the RBD ELISA cannot be the sole method of assessing the platforms, a virus neutralization assay should be included. Please also address the other concerns noted. 

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Benhur Lee

Section Editor

PLOS Pathogens

Benhur Lee

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In this manuscript the authors compare raise in virus neutralizing and binding antibody titers in serum obtained from study participants that had received ChAdOx1-S (AstraZeneca) or BBIBP-CorV (SinoPharm) before followed by a homologous booster vaccination or heterologous vaccination booster with the recombinant PastoCovac vaccine. The outcome of such studies is informative and important since heterologous prime/boost regimes are currently being rolled out as it became clear that not all COVID-19 vaccines that were initially used early during the pandemic had similar vaccine efficacy, and this might be corrected using a heterologous booster strategy. The manuscript is written well however I have some major concerns.

Reviewer #2: Eybpoosh et al report on a study measuring the immunogenicity and safety of a recombinant protein based COVID-19 vaccine (PastoCovac Plus) that was administered as a booster in individuals that had received two doses of ChAdOx1-S or BBIBP-CorV previously. Two additional groups of individuals received a third dose of ChAdOx1-S or BBIBP-CorV. Total Spike specific antibody responses and neutralizing antibody titers were measured. No differences in antibody titers were observed between all four groups, although the highest titer was detected in the heterologous boost groups. The highest fold increase was observed in the BBIBP-CorV + PastoCovac Plus, but this effect is likely caused by the lower antibody response in these individual prior to the boost. Overall, the abstract and the title of the manuscript are supported by the data.

Major comments:

• Please add P-values to the figures to indicate significance (or lack thereof) within and between groups.

• Some individuals were infected during the course of the study and it is not clear if these were excluded or included in the final analysis.

• The neutralization assay is unknown to me and the data seem odd. Is there an upper limit to this assay? What kind of assay is it, a pseudovirus neutralization assay? Is it possible to provide the serum dilutions that resulted in 50% or 100% virus neutralization? The data is very tight at the top and I wonder if you are losing resolution.

• The recombinant protein vaccine can be described a bit more in the manuscript. It is composed of the RBD alone, which can affect the data in Figure 2 where you are measuring the antibody response to the entire Spike protein.

• Line 264 you mention a that "PastoCovac Plus leads to a significant anti-spike IgG mean rise compare to the homologous booster recipients". What is this statement based on? Based on Figure 2, I find that hard to be true for the ChAdOx1-S groups.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: The different vaccine types that study participants have received need to be better described. Along the same line, the results should be discussed in light of the different vaccine platforms that were used (adenovirus, recombinant protein, inactivated virus vaccine + adjuvant).

The potential impact of COVID-19 history is not clearly represented. Which samples in figures 2 and 3 are from individuals with previous COVID-19 exposure. A color coding system might help here.

Why are the antibody titers (both ELISA and neutralization) before boost so different between the homologous and heterologous boosted groups for the BBIBP-CoV groups? The differences between these pre-booster titers are even significant according to Table 2. The fold rise in titers in boost is the heterologous boost group is large but the initial pre-boost titers are very low as well.

Sup. Table 1.: COVID-19 History for the homologous ChadOx1-S: n=13 for Yes and 14 for No. These numbers are the opposite in the other parts of the column for the same samples. Are these numbers swapped by accident?

Since there seems to be COVID-19 history available during and after the study time points, the authors should elaborate more on vaccine efficacy/correlation with observed antibody titers and protection from infection.

Reviewer #2: An ELISA against the RBD alone could be useful and re-analyzing or re-doing the virus neutralization assay

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: none

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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Dear Prof. Ramezani,

We are pleased to inform you that your manuscript 'Immunogenicity and safety of heterologous boost immunization with PastoCovac Plus against COVID-19 in ChAdOx1-S or BBIBP-CorV primed individuals' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Benhur Lee

Section Editor

PLOS Pathogens

Benhur Lee

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: my previous concerns were addressed adequately by the authors

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: NA

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: NA

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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: Michael Schotsaert