We appreciate the editorial staff and the reviewers for taking time to carefully review the manuscript and give detailed and constructive comments, which has greatly helped to improve this paper. Below is our point-by-point response to each comment.

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Response : Thank you reminding us. We have made sure that the manuscript meets the PLOS ONE’s style requirements.

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I have read the journal's policy and the authors of this manuscript have the following competing interests: Novilia Sjafri Bachtiar and Rini Mulia Sari are employees of PT Bio Farma at the time of the conduct of this study and manuscript preparation.

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Response : Thanks for the suggestions. We have added the following statement and revised the competin interests section. (Page 16, Line 338-342)

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Response : Thank you for pointing this out. We have added the caption of our Supporting Information files at the end of the manuscript. (Page 20, Line 418-423)

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Response : Thank you for reminding us. We have removed the institutional logo from the original protocol.

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Response : We have attached the the study sponsor’s permission along the other supporting file.

Review Comments to the Author

Reviewer #1: This manuscript reports immunogenicity and safety of Quadrivalent Influenza HA vaccine compared with Trivalent Influenza HA vaccine and evaluation of Quadrivalent Influenza HA vaccine batch-to-batch consistency in Indonesian children and adults. I have below comments.

Page 5, line 91, “open-labeled” is not consistent with the description in trial protocol where “double-blind” was panned for 9-40 years of age.

Response : Thank you for reminding us. We have revised this part based on the protocol.

Revised manuscript (Page 2, Line 24)

This was an experimental, randomized, double blind, four arm parallel group bridging study involving unprimed healthy children and adults aged 9–40 years. A total of 540 subjects were enrolled in this study and randomized into four arm groups.

Revised manuscript (Page 5, Line 96-98)

This was an experimental, randomized, double blind, four arm parallel group bridging study to assess immune response and safety after one dose of quadrivalent and trivalent influenza HA vaccine in subjects 9-40 years old in clinically health children and adults aged 9–40 years.

Page 6 sample size consideration is not consistent with the description in trial protocol. Please correct and make the descriptions in details about how sample sizes were determined.

Response : Thank you for the suggestion. We have revised the descriptions about how sample sizes were determined clearly.

Revised manuscript (Page 7, Line 140-144)

The sample size was determined based on a 95% confidence interval (CI) and a test power of 80%. The required sample size was 112 in each batches, with the assumption that not all the subjects could complete the study, the total number of subjects was added at least 20% from the minimum requirement (N x 1.2) = 134. Approximately 135 subjects per group will be involved in this study.

Page 7, line 129, (1/1-0.1)*115 cannot obtain 128.

Response : Thank you for pointing this out. We have revised this part.

Revised manuscript (Page 7, Line 141-144)

The required sample size was 112 in each batches, with the assumption that not all the subjects could complete the study, the total number of subjects was added at least 20% from the minimum requirement (N x 1.2) = 134. Approximately 135 subjects per group will be involved in this study.

Page 7, line 133, please make it clear which data analysis will use these mentioned statistical tests.

Response : We have revised this part to make it clear.

Revised manuscript (Page 7, Line 148-153)

Demographic data were expressed as mean, standard deviation, and range values. Analysis of Geometric Mean Titer (GMT), seroprotection, and seroconversion rates between the vaccine groups was performed using Kruskal-Wallis and Chi-square or Kolmogorov-Smirnov tests. The differences of antibody concentration for each antigen before and after primary series of Influenza vaccine was analysed using Wilcoxon test. Values of p < 0.05 indicated statistically significant differences between groups were assessed by Chi-square test. Immunogenicity analyses were performed on the per protocol population. Safety data were analysed in the intention to treat population.

Page 9, Table 2, Mann-Whitney test is not suitable to test count or proportion data. Chi-square may be used.

Response : We apologize for the mistake. For table 2, we used chi-square test for analyzed the data and we have revised the explanation. (Table 2; Page 9, Line 185-186)

Please tell what statistical test was used for Fig. 2.

Response : For figure 2, we used mann whitney test as the statistical test (Figure 2)

Reviewer #2: This was an open-labeled, bridging clinical study to assess the immunogenicity and safety of QIV compared with TIV and to evaluate batch-to-batch consistency in three consecutive batches of QIV. As a solid, well-performed clinical trial, this manuscript will be an important addition to the literature, however, there are some points that the authors should address that can improve the manuscript.

Major comments

1. The title of this manuscript includes “evaluation of Quadrivalent Influenza HA vaccine batch-to-batch consistency in Indonesian children and adults”, however, the results of this part are not described in the conclusion of the abstract.(Page3, Line38-40)

Response : Thank you for the comments. We have added the results of the “methods and findings” part in the abstract to describe the title. And in the “conclusion” of the abstract, there has been already a part which describes the title.

Revised manuscript (Page 2, Line 38-39)

This was an open-labeled, bridging clinical study involving unprimed healthy children and adults aged 9–40 years. A total of 540 subjects were enrolled in this study and randomized into four arm groups. Each subject received one dose of TIV or QIV with three different batch codes. Serology tests were performed at baseline and 28 days after vaccination. Hemagglutination inhibition (HI) antibody titers were analyzed for Geometric Mean Titer (GMT), seroprotection, and seroconversion rates. Solicited, unsolicited, and serious adverse events were observed up to 28 days after vaccination. A total of 537 subjects completed the study per protocol and were analyzed for immunogenicity criteria. All randomized subjects were analyzed for safety criteria. The percentage of the subjects with anti-HI titer ≥1:40 28 days after QIV vaccination was 99.5% for A/H1N1; 99.5% for A/H3N2; 93.1% for B/Texas, and 99.0% for B/Phuket. The seroprotection, GMT, and seroconversion rates of QIV were not significantly different from those of TIV for the common vaccine strains (p > 0.01) and were significantly different from those of TIV for the added B/Phuket strains (p < 0.01). Most solicited injection-site and systemic reactions with either vaccine were mild to moderate and resolved within a few days. Antibody response to QIV were equivalence among vaccine batches and comparable between age groups for each of the 4 strains.

2. “This was the first QIV study conducted by Bio Farma on subjects aged 9-40 years in Indonesia.”, the sentence here does not connect the context and the reference here is the first study on QIV, which is a supplement to QIA study. I think it should be described in detail in the text to increase the rigor of the article.(Page5, Line83-84)

Response : Thank you for pointin out this term. We have revised the sentences in this paraghraph.

Revised manuscript (Page 5, Line 84-90)

At first, Bio Farma formulated TIVs and have conducted several studies on seasonal TIVs between 2008 and 2014. The results of these studies were consistent. TIVs were well-tolerated and induced high antibody titer against influenza antigens and no serious adverse events (AEs) during the study [9–13]. In 2016, Bio Farma starts to formulate the quadrivalent inactivated influenza vaccine that could potentially provide wider protection against influenza B viruses. Several countries has been conducted the studies using Influenza HA Vaccine (quadrivalent vaccine), but none from Indonesia [8,14,15]. This was the first QIV study conducted by Bio Farma on subjects aged 9–40 years in Indonesia .

3. The content about study design is too concise to figure out, this part should be expanded.(Page5, Line89)

Response : We have expanded the explanation of study design part. (Page 5, Line 95-104)

4. Please also fully list inclusion/exclusion criteria in the main text, and confirm these have been unchanged from the protocol.(Page5, Line101-106)

Response : Thank you for the suggestions. We have added the inclusion and exclusion criteria in main text based on the protocol

Revised manuscript (Page 6, Line 108-120)

The primary inclusion criteria included healthy children and adults aged 9–40 years who have signed the informed consent form and committed to complying with study instructions and trial schedules. Subjects were not eligible if the subject have enrolled or scheduled to be enrolled in another trial, presented with mild, moderate, or severe illness with a fever (axillary temperature ≥37.5⁰C), history of allergy to egg, chicken protein, or other vaccine, uncontrolled coagulopathy or blood disorders contraindicating intramuscular injection, received in the previous 4 weeks a treatment likely to alter the immune response [intravenous immunoglobulins, blood-derived products, or long-term corticosteroid therapy (>2 weeks)], pregnancy and lactation (adult), have any abnormality or chronic disease which according to the investigator might interfere with the assessment of the trial objectives, have already immunized with influenza vaccine within 1 year or any vaccination within 1 month before and after immunization of Quadrivalent Influenza Vaccine.

5. Please state clearly, in the methods what were the primary and secondary endpoints of the study.(Page6, Line125)

Response : Thank you for the suggestions. We have added the primary and secondary endpoints in “Outcomes” part in Methods section.

Revised manuscript (Page 8, Line 165-171)

The primary outcome was to evaluate the percentage of subjects with anti-HI titer � 1:40, 28 days after Influeza HA vaccination. The secondary outcome was to evaluate geometric mean titres, percentage of subjects with increasing antibody titer � 4 times and/or percentage of subjects with transition of seronegative to seropositive between one dose of quadrivalent and trivalent influenza HA vaccine in subjects 9-40 years old and between each batch number of Quadrivalent Influenza HA vaccine and the incidence rate and intensity of adverse events or any serious adverse events within 30 minutes, 72 hour and 28 days after immunization.

6. Table 4 as the main table should be described in the text.(Page11, Line183)

Response : Thank you for reminding us. We have added the explanation of table 4 in the text.

Revised manuscript (Page 10, Line 213-216)

Batch-to-batch equivalence for each strain was concluded if the two-sided 95% CI of each strain GMT ratio of the compared batches was between 0.67 and 1.5 (Table 4) [14]. The GMT ratios of the overall post-vaccination GMTs for each pair of lots for each strain were all between 0.67 and 1.5.

7. If the p value is <0.0001, please indicate it in the figure.(Figure 2)

Response : Thank you for the suggestion. We have added the p value (<0.001) in figure 2 (day 28, B/Phuket)

Minor comments

1. Materials and methods: “Sample size and study analysis” is best described in two parts.(Page6, Line125)

Response : Thank you for the suggestions. We have described those parts separately. (Sample Size; Page 7, Line 139-144 and Study analysis; Page 7-8, Line 145-171)

2. Line 101-106, please carefully check the inclusion and exclusion criteria are consistent with the protocol.

Response : Thank you for pointing this out. We have added the inclusion and exclusion criteria based on the protocol.

Revised manuscript (Page 6, Line 108-120)

The primary inclusion criteria included healthy children and adults aged 9–40 years who have signed the informed consent form and committed to complying with study instructions and trial schedules. Subjects were not eligible if the subject have enrolled or scheduled to be enrolled in another trial, presented with mild, moderate, or severe illness with a fever (axillary temperature ≥37.5⁰C), history of allergy to egg, chicken protein, or other vaccine, uncontrolled coagulopathy or blood disorders contraindicating intramuscular injection, received in the previous 4 weeks a treatment likely to alter the immune response [intravenous immunoglobulins, blood-derived products, or long-term corticosteroid therapy (>2 weeks)], pregnancy and lactation (adult), have any abnormality or chronic disease which according to the investigator might interfere with the assessment of the trial objectives, have already immunized with influenza vaccine within 1 year or any vaccination within 1 month before and after immunization of Quadrivalent Influenza Vaccine.

3. Line 146, the results of this manuscript are poorly described, and the author is advised to restate it. In particular, important results should be detailed.

Response : Thanks to the reviewer for pointing out this part. We have re-described the results section and have pointed out the important results. (Line 173)

4. In figure 1, QIV Batch A, “exluded from analysis (Protocol noncompliant, n=134)” should be “excluded” and “n=1”.

Response : Thanks for the comment. We have revised the description in figure 1. (Figure 1)

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Response : Thanks for the chance. We agree to publish the peer review history for our manuscript.

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Response : Thanks to the reviewer for pointing out this part. We already converted Figure Files to PACE.

Attachments

Attachment

Submitted filename: Response to Reviewers.docx

PONE-D-22-35728R1

Immunogenicity and safety of Quadrivalent Influenza HA vaccine compared with Trivalent Influenza HA vaccine and evaluation of Quadrivalent Influenza HA vaccine batch-to-batch consistency in Indonesian children and adults

PLOS ONE

Dear editorial staff and the reviewers,

We appreciate the editorial staff and the reviewers for taking time to carefully review the manuscript and give detailed and constructive comments, which has greatly helped to improve this paper. Below is our point-by-point response to each comment.

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Response : Thank you for reminding us. We have reviewed our reference list, and we have changed some cite and references which couldn’t be accessed anymore to the new one.

Reference no. 7

World Health Ogranization. Recommended composition of influenza virus vaccines for use in the 2013-2014 northern hemisphere influenza season. February 2013. Available from: https://www.who.int/publications/m/item/recommended-composition-of-influenza-virus-vaccines-for-use-in-the-2013-2014-northern-hemisphere-influenza-season (accessed June 12, 2023).

Reference no. 13

Luo FJ, Yang LQ, Ai X, Bai YH, Wu J, Li SM, et al. Immunogenicity and safety of three 2010-2011 seasonal trivalent influenza vaccines in Chinese toddlers, children and older adults. Hum Vaccin Immunother. 2013 Aug 1; 9(8): 1725–1734. doi: 10.4161/hv.24832.

Reference no. 16

Food and Drug Administration Center. Clinical Data Needed to Support the Licensure of Seasonal Inactivated Influenza Vaccines. Guidance for Industry. 2007: 1-16. Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/clinical-data-needed-support-licensure-seasonal-inactivated-influenza-vaccines. (accessed June 12, 2023)

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.

Response : Thank you for the comment. We have adequately addressed our comments raised in the previous round of review.

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.

Response : Yes, our manuscript has already described a technically sound and the data has already supported the conclusions

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

Response : Yes, the statistical analysis has been already performed appropriately and rigorously.

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.

Response : Thank you for reminding us. We have added one table (Table 3. Geometric Mean Titers (GMT) to influenza A (H1N1), A (H3N2), and B virus strains Pre- and 28 days post-immunization) to explain the result in page 9, line 187-192.

Revised manuscript (Page 9, line 185-190)

Comparison of GMT between subjects who received QIV and TIV was shown on table 3 and figure 2. No significant differences in GMT 28 days after immunization between one dose of QIV and TIV in the 9–40-year-old subjects for A/California/7/2009 (X-179A) (H1N1) pdm09, A/Hong Kong/4801/2014 (X-263) (H3N2), and B/Texas/2/2013 (p = 0.322, p = 0.536, and p = 0.378, respectively). However, in strain B/Phuket/3073/2013, GMT 28 days after immunization was significantly higher in QIV group than in TIV group (p < 0.001).

Table 3. Geometric Mean Titers (GMT) to influenza A (H1N1), A (H3N2), and B virus strains Pre- and 28 days post-immunization

Fig 2. Hemagglutination inhibition (HI) Geometric Mean Titers (GMT) before and 28 days after QIV and TIV immunization.

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.

Response : We have already made sure that language we used in this manuscript have been clear, correct and unambiguous.

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 #1: Line 149-150, “The 149 differences of antibody concentration for each antigen before and after primary series of Influenza vaccine was analysed using Wilcoxon test”. Please indicate clearly which Wilcoxon test was used.

Response : In this manuscript, there is no data which used Wilcoxon test to analyse the data. We have deleted this part. (Page 7, line 146-151)

Line 150-151, “Values of p < 0.05 indicated statistically significant differences between groups were assessed by Chi-square test”. This sentence is not clear. Why was this cut-off p-value only used for analysis assessed by Chi-square test? You may want to say “Values of p < 0.05 indicated statistically significant differences between groups.”

Response : Thank you for the suggestion. Yes, we have revised this line as suggested. (Page 7, line 149)

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.

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Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

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Submitted filename: Response ro Reviewers.docx