PONE-D-22-17990Comparison of vaccination efficacy using live or UV-inactivated influenza viruses introduced by different routes in a mouse modelPLOS ONE

Dear Dr. Kwon,

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 Dear Dr. Hyung-Joo Kwon, Your paper has been reviewed by two experts in the field who state the significant of the studies presented in your paper. However, both reviewers cited a number of weaknesses that need to be adequately addressed. It would be especially important to address the comments by reviewer 2 who has some significant concerns about some technical aspects of the work. Please submit your revised manuscript by Sep 22 2022 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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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

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: Kyeongbin Baek et al. investigated the protective effects of live and UV-inactivated

influenza viruses via intraperitoneal and intramuscular administration. Cross-

protection against different subtypes of influenza virus was also investigated. The

compositions and administration routes of vaccination are important issue to

consider in order to enhance the efficacy of vaccines. In the case of the UV-

inactivated A/WSN/1993 (H1N1) virus, only a high dose of intraperitoneal

administration induced some degree of protection against intranasal challenge with

live A/WSN/1993 virus. However, low dose of live A/WSN/1993 virus via both routes

of administration showed complete protective effects. Intraperitoneal or

intramuscular administration with live- or UV-inactivated A/Philippines/2/1982 (H3N2)

virus produced no cross-protective antibodies, and resulted in no cross-protection

against intranasal challenge with A/WSN/1993. This information can be used as a

practical data in order to develop an effective influenza vaccine for cross-protection.

The experiments are well conducted with appropriate data analysis. Therefore, I

recommend the manuscript to be accepted for publication, while it is necessary to

finish the revision as follows:

1. The authors used ELISA to measure the amount of influenza A virus-specific

IgG. It will be necessary to describe whether the WSN or H3N2 Php virus

coated on 96-well plates is a live virus or a UV-inactivated virus.

2. In Figure 5, cross-reactive IgG was produced by intramuscular administration

of live viruses. However, cross-protection against different subtypes of

influenza virus was not observed. Could you add some more discussion with

the concept of threshold?

3. On line 316-318, there is a sentence ‘‘Additionally, the cytokine levels in sera

and lungs were not different than those in uninfected mice when the mice

were intranasally challenged with live WSN 14 days after intraperitoneal

inoculation with live H3N2 Php (data not shown).’’ Please show the results in

the supplementary data.

Reviewer #2: Here, Baek et al. reported a study on evaluating the impact of vaccination administration routes on the vaccine efficacy using both inactivated and live influenza viruses. They compared the Intramuscular and intraperitoneal inoculation. They assessed antibody responses (ELISA) and protective immune responses (viral lethal challenge study). Moreover, they tested the breath of immune responses induced by these routes of vaccination. They found that there was a protective immunity induced by either route of vaccination but only when a high does was used. Finally, they tested the breath of immune responses triggered by their immunization.

Overall, this manuscript targeted on an important topic, the impact of vaccination route on efficacy. The entire story will benefit from a more stringent experimental design, including critical information and more precise description.

Major points:

1. Given that they used IM and IP as their vaccination route, they induced humoral responses through systematic immune responses. Thus, cellular immunity should play an important role in regulating the humoral responses. To strengthen their conclusion, it is important to provide the audience some experimental information about cellular immune responses. Particularly, they tested the immune responses towards a heterologous virus. This is likely because of cellular responses towards the internal genes, which are more conserved between the viruses they tested. Therefore, it is important to present data about T cells responses after their vaccination.

2. In the manuscript, they evaluated the antibody by ELISA. This only gave the information about the level of specific antibodies, but not potency for those specific antibodies. HI or microneutralization assay should be adequate to address this concern.

3. The manuscript will benefit from a more precise description. For example, the live influenza virus used here is not a live attenuated vaccine. Another example would be the description of dosage used for challenge. Instead of using specific PFU, a much more meaningful way would use an equivalent MLD50 value. In such a way, the readers will be better informed about their challenge stringency.

Minor points:

1. Line 28, it is a typo for “Influenza A/WSN/1993”

2. There is a lack of consistency in experimental design. For example, in Figure 3, in this set of in vivo studies, they only observed animals 7 days post challenge. In their other in vivo studies, they observed animal 10 days post challenge.

3. The amount of protein (ug) would be a more proper unit for describing the dosage used for IM inoculation.

4. There is no indication to authenticate their inactivation.

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Reviewer #1: No

Reviewer #2: No

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Comparison of vaccination efficacy using live or ultraviolet-inactivated influenza viruses introduced by different routes in a mouse model

PONE-D-22-17990R1

Dear Dr. Kwon,

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.

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Kind regards,

Juan Carlos de la Torre, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

In this revised version of their paper, the authors have adequately addressed most of the comments and concerns raised by the reviewers.

An issue that was not addressed relates to the T cell responses. However, the authors have provided a reasonable argument about the reasons why these data have not been incorporated into the revised version of their paper.

Reviewers' comments: