Dear Dr. Ross,

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Additional Editor Comments:

Please find a number of comments to your paper, almost all of which are aimed to improve its clarity. Please carefully review and respond to these.

Major comments:

It’s difficult for an observational study with a huge amount of data but try to emphasize what the questions were that the study hoped to answer and whether it could answer them. For example, how do potential vaccines for elderly compare in antibody generating capacity? Limitation: Flublok not used in elderly.

Example: what is the effect of skipping years? There might be better responses after a skipped year, but also more vulnerability during that skipped year. (recognizing that a study such as this could not provide sufficient data to call into question a standing recommendation.)

It seems that the data describing the tables and graphs is in the text rather than in Legends/Footnotes. Each table and graph should be able to stand alone and be interpretable. Sometimes, they are not clearly enough labelled to do so.

Please clarify as much as possible in Table/Figure and text when data relate to the repeater cohort and when to the overall population.

Minor comments

Abstract:

Clarify whether the lower titers in elderly people were in fact a factor in the calculated higher seroconversion rates, i.e. by allowing individuals to regain seronegative status prior to the next year’s vaccination, were more seroconversions (which require seronegativity??), made possible?

For the Flublok vs Hi dose FluZone comparison, state whether ages were comparable in the 2 groups. An age disparity would be a limitation on comparing the vaccines.

Line 46-8 “…serum HAI activity was similar in elderly participants 47 following vaccination with either Fluzone HD or Fluad against both influenza A or B vaccine 48 components”. Please clarify what is being compared here; is it FluA and FluB titers after the two vaccines mentioned? Or is there a broader comparison here?

Methods. No vaccine in the 4 weeks since last vaccination seems an unusual criterion, since people of this age would not be expected to receive two influenza vaccines only 4 weeks apart. Is there some rationale that could be explained?

Table 2 Footnote. IAV (H1N1) strains are selected to reflect circulating (missing word?),

Table 3. If an individual in the repeater cohort moved from one age category to the next over 3 years, how were the data handled? Is Table 3 the original age or is the actual age in that year used?

Line 210. In discussion, consider effect of overall greater age?

Line 212. The proportion of adolescents (≤17 years) was relatively small in the 2022-2023 season (9.5%) 213 and 2024-2025 season (5.3%) but increased to 6.5% in 2023-2024 season. The text seems to imply the last season was greater than the first, but in fact first season is greatest. Consider rewording?

Line 239. Consider stating ages receiving FluMist, and whether recipients had previously been vaccinated with IAV. Prior HAI titer might interfere with take.

Question: were all vaccinations given according to manufacturers age recommendations/indications?

FIGURE 1

Needs a legend, which I did not find. Is it correct that the first column is D0 and the second D28 under each strain?

FIGURE 2

It’s not easy to follow the rise in titer within each age group for each year. The lines connect baseline to baseline and post-vaccination to post-vaccination, which does not seem like the primary focus. Perhaps all values could be on a 0, 28 day timeline with before and after vaccination linked by a line. Then the confidence intervals could be displayed.

Line 242. “Table in S1 Table summarizes” wording is awkward

Line 270 “had similar baseline HAI 270 titers from participants” perhaps say similar TO?

Line 263. The heat map is a good idea. Shouldn’t this explanation be, at least in part, in the legend? What does the blue column on th right (H3N2) mean? It appears to mean participants are ordered by decreasing titer, but they don’t seem to be.

LINE 285. “ Fewer participants between 18–34 years old had HAI titers >1:40 at D0 during the 2022-2023 season compared the 287 next two seasons, but had HAI titers >1:40 following vaccination on D28, with modest back288 boosting against historical H3N2 isolates. The point of the second part of the sentence is not clear—is a word missing??

Line 480 and following and Table 4. Is this referring only to the final season of the study? How do 2017-2019 come into it (footnote to Table 4)? Line 485…which participants are meant by “these participants”? Line 490 and following is very hard to follow. Should it say …participants WHO were not……? Is the B Victoria component of the vaccine influencing the B Yamagata response? Please comment in Discussion section.

Line 530… those who switched from SD to…word(s) missing ???

It’s hard at first to see what’s different between two paragraphs in results describing Figs 6 and 7, and the two figures. Titles emphasizing IAV and IBV would help.

Line 668-671. ….’a smaller proportion had detectable titers and achieved…. “ Possibly this could be more clearly stated as ‘ a smaller proportion fell into the group that initially had detectable titers and then achieved..”

Line 714. A D amino acid sounds like chirality is being discussed. ‘..had the amino acid D’ might be clearer.

Is it correct that titers to B Yamagata rose during the 2024-25 season even though it was not in the vaccine? Could this be because of a few outliers who got infected with B Yamagata and raised the average or extreme cross-boosting?

Line 737 ‘and thereby avoidS…

Line 738… while this statement (may avoid mismatches with circulating strains due to egg adaptation) may be correct, does it explain your results? Methods states you grew the indicator viruses for the HAI assays in eggs? (Was that a disadvantage for FluBlok and FluCelVax measured titers? Please comment.

735 and following—please clarify whether your data sheds any light on the relative value of Flublok vs FluAd and/or Fluzone HD in older adults.

748 and following—it might be worth looking at titers in pediatric participants alone. Also, perhaps mention that half the age span of pediatric patients in general was omitted from this study. Younger children’s responses probably differ.

Line 762. “In contrast, participants in all age groups had (descriptive word missing?) HAI titers against the two influenza B vaccine components compared to HAI titers against influenza A vaccine components.”

Reviewers' comments:

Comments to the Author

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

Reviewer #1: Yes

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

Reviewer #1: I Don't Know

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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

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

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Reviewer #1:  Reviewer Comments: Reviewer Comments:

Manuscript Number: PONE-D-25-48769

Title: Assessment of Hemagglutinin-Inhibition Activity Following Influenza Vaccination During the 2022-2023, 2023-2024, and 2024-2025 Seasons.

This is a 3-season report on the immunogenicity of six influenza vaccine types in 1328 participants aged 9-89 years old enrolled in an observational cohort study, 203 of whom received an influenza vaccine in all 3 seasons allowing longitudinal assessment. Overall, the manuscript is well written, however there are a few major and multiple minor revisions needed.

Major Comments:

1. Page 21, Lines 165-167: HAI titers of ≥40 are considered non-detectable or seronegative in this manuscript, which is incorrect as the lower limit of detection of HAI test is a dilution of 1:10, and <10 is considered not detectable or seronegative. Also, a titer of ≥40 or more is considered seropositive or seroprotection (if it is a post-vaccination titer), but seroprotection and seropositivity are used interchangeably on D0 in the figures. Consider using alternative terminology such as no/low HAI titer <40 vs. HAI titer ≥40. Other alternatives are using no-seroprotection vs. seroprotection. The definition of seroconversion looks good.

2. Page 22, Line 195: Regarding comparisons between vaccine types, Dunnett’s multiple comparison test is appropriate if there is one control group and the one-way ANOVA is significant. If there are multiple comparison groups, then Tukey or Scheffe test may be more appropriate.

3. Page 24, Line 245: Supplemental Table 1 and text have discrepant vaccine types – e.g. line 245 (FB>FB>FB; n=16) whereas Supplemental Table 1 shows FM>FM>FM, n=16 and does not show anyone with FB>FB>FB. Also, this Table shows FB > FC > FB = 8 in row 6 from top and again FB > FC > FB = 1 four rows up from the bottom Total row. Similarly, FZ SD >FZ HD > FZ HD are listed twice with N = 5 and 1 each.

Minor Comments:

The manuscript needs correction or clarification at multiple places including in the tables and figures.

1. Page 11, Line 59: Please clarify if the lower vaccine-induced response is meant for standard-dose inactivated vaccine.

2. Page 12, Line 89: Please clarify if individual vaccinees with more than one dose in a study season were excluded or not.

3. Page 19, Line 121, Table 3: Please clarify with a footnote when the age group and BMI are for individuals in Table 3. ?First vaccination, last vaccination or some other time.

4. Page 21, Line 173. Please spell out what BEI stands for.

5. Page 23, Lines 212-213: The proportion of adolescents (≤17 years) was relatively small in the 2022-2023 season (9.5%) and 2024-2025 season (5.3%) but increased to 6.5% in 2023-2024 season. Please correct this sentence because the proportion of children <18 years decreased from 9.5% to 6.5% to 5.3% in the 3 consecutive study seasons.

6. Page 23, Lines 216-218: Consider deleting this sentence here because supporting data is shown later under vaccination patterns in repeaters.

7. Page 25, line 267: Figure 1A Y-axis label shows ages 10-17 years instead of 9-17 years.

8. Page 25, line 276: There is no titer done at 6 months from the pre-vaccination baseline so it might be better to say that the titer had waned prior to vaccination next season rather than wanes during the season.

9. Page 25, lines 283- 285: There were no titers done 365 days after 2024-25 season and DR21 titers seem to have waned by 2024-25 baseline, please clarify this sentence.

10. Page 28, line 361: Statistically “similar” or “significant”?

11. Page 28, line 378: Clarify seroconverted against A (H1N1).

12. Page 28, line 384: Is the 28% B/Yamagata seroconversion correct for average over 2 seasons and is it truly the lowest? Is it lower than 31% average seroconversion for A (H1N1) over 3 seasons because average B/Yamagata seroconversion needs to be assessed over first 2 seasons only.

13. Page 29, Figure 4, line 401. There are no numbers seen on the X-axis in this figure, it shows the seasons.

14. Page 30, line 418: Re ages 18-34 years, should this be 35-49 years?

15. Page 30, line 420: Re 33-53% against A (H3N2)? Please clarify.

16. Page 30, lines 428-429, re B/Vic: Oldest participants show higher seroconversion than youngest in 2 of 3 seasons.

17. Page 30, lines 434-435: Number of participants not seen along the X-axis in Fig 5.

18. Page 30, lines 434-435: Consider replacing UGA9 with 2024-2025 season.

19. Page 32, Table 4, line 478: Seasons listed need corrections.

20. Page 32, Table 4, line 479: It is not clear if the Fisher’s exact test was used for this table as results are not shown or described in the text until the discussion – please move this to the results or table.

21. Page 33, line 493: Correction: 30% did seroconvert. Delete not at the end of the sentence.

22. Page 35, line 556, consider naming the six vaccines here.

23. Page 36, line 588: Correction - 2023-2024 season (Fig 9B) compared to 202-23 season (Fig 9A).

24. Page 37, Fig 9, line 619: Red Asterix is not seen.

25. Page 38, line 645 – correction, to historical strains rather than to drifted viral variants.

26. Page 39, line 662, consider saying to previous two years rather than 1-2 years.

27. Page 39, line 669, change detectable to protective or whatever alternative terminology is used, as appropriate.

28. Page 41, line 735, specify participants age range for Flublock

29. Page 42, line 770: to maintain ‘seroprotection or HAI titers ≥40’.

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Assessment of Hemagglutinin-Inhibition Activity Following Influenza Vaccination During the 2022-2023, 2023-2024, and 2024-2025 Seasons

PONE-D-25-48769R1

Dear Dr. Ross:

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Academic Editor

PLOS One

Additional Editor Comments (optional):

Thank you for your careful attention to the reviews.

Reviewers' comments: