PONE-D-24-30084A Single Dose of Inactivated Influenza Virus Vaccine Expressing COBRA Hemagglutinin Elicits Broadly-Reactive and Long-Lasting ProtectionPLOS ONE

Dear Dr. Ross,

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Academic Editor

PLOS ONE

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2. Thank you for stating the following financial disclosure:

“This project has been funded as part of the Collaborative Influenza Vaccine Innovations Centers (CIVICs) by the National Institute of Allergy and Infectious Diseases, a component of the NIH, Department of Health and Human Services, under contract 75N93019C00052.  TMR is also supported in part as a Georgia Eminent Scholar by the Georgia Research Alliance, GRA-001.”

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

“We would like to thank James Allen, Ivette Nuñez, and Ying Huang for designing COBRA HA vaccines and Spencer Pierce for purifying the HA antigens. We thank Naoko Uno technical assistance. We also thank the University of Georgia Animal Resource staff, technicians, and veterinarians for their excellent animal care. We also appreciate Benjamin Chadwick for proofreading and language editing for this paper. This project has been funded as part of the Collaborative Influenza Vaccine Innovations Centers (CIVICs) by the National Institute of Allergy and Infectious Diseases, a component of the NIH, Department of Health and Human Services, under contract 75N93019C00052.  TMR is also supported in part as a Georgia Eminent Scholar by the Georgia Research Alliance, GRA-001.”

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“This project has been funded as part of the Collaborative Influenza Vaccine Innovations Centers (CIVICs) by the National Institute of Allergy and Infectious Diseases, a component of the NIH, Department of Health and Human Services, under contract 75N93019C00052.  TMR is also supported in part as a Georgia Eminent Scholar by the Georgia Research Alliance, GRA-001.”

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: No

Reviewer #2: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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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

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Reviewer #1: The authors report on the immunogenicity and protective potential of WIV and SIV preparations, adjuvanted or not, of computationally optimized broadly reactive antigen preparations in H1N1 and H3N2 infection primed ferrets. In general the WIV preparations protect the animals better than SIV preparations against CA/09 challenge (direct or through contact transmission).

Major remarks:

1. Line 131: the authors should state the scale of the MDCK cultures that were used for the COBRA vaccine virus preparations and whether their HA was sequence verified to exclude any cell culture adaptation of the COBRA-based viruses.

2. Line 168 and figure 1: correct that all ferrets were infected with a mixture of Sing86 and Pan99 viruses in week -8? Please clarify. Also, please provide the outcome of the seroconversion analysis against the challenge viruses in week-6 serum samples. The timeline in Figure 1 should be scaled proportionally and the figure should be adapted such that it is clear that naïve as well as mixed (?) infection primed ferrets were vaccinated in week 0.

3. Figure 3: serum from mock vaccinated ferrets shows MN activity against HK/19, SA/19, and TAS/20. Please clarify. Where these ferrets primed by infection in week -8 and subsequently mock-vaccinated? If so, this should be made clear in figure 1. Correct then that there were 2 control groups: infection primed followed by mock (PBS) vaccination and naïve (mock infected) followed by mock (PBS) vaccination?

4. Figures 5-9: control groups are confusing: naïve and naïve-mock were differently treated? Infection primed only ferrets included?

5. Figure 5 and 8 should specify the number of animals in each group.

Other remarks:

1. Line 48: please check the wording: “… influenza vaccine aims to develop a vaccine …” does not sound right.

2. Line 91: “immunogenicity” instead of “antigenicity”?

3. Line 138: please define BPL.

4. Please provide the source of BioBeads (line 150).

5. Line 353: prime vaccination implies that there was a boost vaccination as well. Please adapt.

6. Line 380 and 392: duplicated figure 3 legend, please adapt.

Reviewer #2: The manuscript describes the in vivo studies on COBRA antigens for influenza in ferrets. Overall, the data provide a substantive and well-supported evaluation of the candidate vaccines, and this review found the transmission experiments to add quite a bit of novelty. A few suggestions are provided to improve the ms:

1. It appears the vaccine groups conferred a trend toward protection from mortality and morbidity relative to mock control, but the differences don't appear to be statistically significant in the survival graphs. Please indicate this with "ns" notation near the mock control.

2. Table 2 has a lot of information and I wonder if it can be simplified to the key points relevant to the text, or perhaps even moved to the supplement.

3. It is not clear how the authors were able to specifically determine HA-stem antibodies in Figure 4. How do you know these titers reflect only stem-binding antibodies? Please explain the rationale in the main text.

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Reviewer #1: No

Reviewer #2: No

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A Single Dose of Inactivated Influenza Virus Vaccine Expressing COBRA Hemagglutinin Elicits Broadly-Reactive and Long-Lasting Protection

PONE-D-24-30084R1

Dear Dr. Ross,

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Kind regards,

Victor C Huber

Academic Editor

PLOS ONE

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