PONE-D-21-10237

The Spike-specific IgA in milk commonly elicited after SARS-CoV-2 infection is concurrent with a robust secretory antibody response, exhibits neutralization potency strongly correlated with IgA binding, and is highly durable over time

PLOS ONE

Dear Dr. Powell,

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Academic Editor

PLOS ONE

Journal Requirements:

1) 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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To comply with PLOS ONE submission guidelines, in your Methods section, please provide a more detailed description of your methodology, specifically for assays measuring IgA, IgG, and secretory-type Abs. Please ensure that you describe the sources and catalog numbers (if applicable) of all kits, proteins, antibodies, equipment, etc. in the methods section of your manuscript.

5) In your Methods section, please provide additional information about the participant recruitment method and the demographic details of your participants. Please ensure you have provided sufficient details to replicate the analyses such as:

a) the recruitment date range (month and year),

b) where patients were recruited from,

c) a table of relevant demographic details,

d) a statement as to whether your sample can be considered representative of a larger population, and

e) a description of how participants were recruited.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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.

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: No

Reviewer #2: Yes

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4. 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.

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: This manuscript reported that secretory IgA response was dominant among the human milk samples and the duration of IgA was 4-10 months. The authors also observed that IgA binding (ELISA) and neutralization capacities (spike-pseudotyped VSV) were positively correlated. These findings have already reported in previous studies. The authors have confirmed the previous studies but the new key findings that increase the knowledge in the field of SARS-CoV-2-specific antibodies in human milk are missed in this study.

-Pace RM, Williams JE, Järvinen KM, Belfort MB, Pace CD, Lackey KA, Gogel AC, Nguyen-Contant P, Kanagaiah P, Fitzgerald T, Ferri R. Characterization of SARS-CoV-2 RNA, antibodies, and neutralizing capacity in milk produced by women with COVID-19. Mbio. 2021 Feb 23;12(1).

-Demers-Mathieu V, DaPra C, Mathijssen GB, Medo E. Previous viral symptoms and individual mothers influenced the leveled duration of human milk antibodies cross-reactive to S1 and S2 subunits from SARS-CoV-2, HCoV-229E, and HCoV-OC43. Journal of Perinatology. 2021 Mar 1:1-9.

Concern about this manuscript:

Abstract

1. The title and abstract are extremely long (PLOS One: 250 characters for title). Authors should summarize the most important findings for the abstracts (PLOS One: 300 word max). In most of peer-reviewed Journals, references are not present in the abstract.

2. “antibodies bearing secretory component” seem a strange and not appropriate term to describe secretory antibodies (line 38).

Background

1. Infants younger than 6 months of postnatal age, cannot be vaccinated due to their immature immune system. It is why influenza and pertussis vaccines are given after 6 months.

2. Mistake “ia” should be “is” (line 74)

Study participants

1. The criteria of inclusion and exclusion for the donors are missing in the methods.

2. How the authors determined the sample size?

3. Do all donors had a clinical /instrumental diagnosis of COVID-19 infection (COVID-19 PCR test)? This information should be presented in the method.

4. Donors and Control groups are not adequately described. All the clinical characteristics of the participants are missing. The demographic description is critical as these maternal factors influence the breast milk antibody titers and neutralizing activity between mothers.

Analytical Methods

1. Control negative with only media and control negative with milk with low/absent SIgA activity (heat-treated human milk) were performed as controls in the experiment? These controls are critical as human milk contains other antimicrobial components that could reduce the viral infectivity.

2. How the sample size calculation was done to obtain good power?

Discussion

1. Authors should explain why they selected 4-6 weeks and 4-10 months post-infection to evaluate antibody titers and neutralizing activity.

2. Limitations of this study is missing and should be included.

Figure.

1. Asterisk to show difference between groups are missing on all Figures.

2. Authors should also add the statistical analysis in the figure legend and the sample size of each group.

Table

Add a table with the demographic description of the participant

Reviewer #2: Summary

In the submitted manuscript, Fox et al report on the milk antibody response specific to the SARS-CoV-2 spike protein following maternal infection. This report greatly expands on the group’s previous report by recruiting a larger cohort of individuals (n=75 participants) and collection of milk samples at 4-6 weeks and 4-10 months post infection. This study provides valuable data on the longer-term durability of the milk antibody response following maternal COVID-19. Further, it also expands on other studies that have examined the ability of human milk to neutralize SARS-CoV-2 by isolating IgA from milk and demonstrating it to be a key factor of milk that effectively neutralizes SARS-CoV-2. This work is of both biological and clinical significance.

Abstract

Per journal guidelines, the abstract should not contain citations. Some of these references are never mentioned in the Introduction (e.g., refs 3-5) and I would suggest including them there.

The concluding sentence is very long and should be restated.

Introduction

Line 63 – should “previously-infected” be hyphenated?

Line 74 – “ia” – typo

Line 71 – “Notably, after two hours in the infant stomach, the total IgA concentration decreases by <50%, while IgG concentration decreases by >75%; importantly, though total SC concentration decreases by ~60%, there ia no decrease in the stomach of infants born pre-term (within the first 3 months of life) – a population highly vulnerable to infection” – This sentence is confusing as it appears to be discussing decreased in IgA, but then ends with stating that there is no decrease in preterm infants (as measured during the first 3 months of life). The reference cited seems to indicate that there is a larger decrease in IgA in preterm infants compared to term infants, but IgG and IgM appear more stable. Please clarify.

Methods

Were any of the participants/data from the group’s previous publication included in this work, if so, can you include that information here. More details should be provided on where (general region is fine) and when sample collections took place, per IRB stipulations.

Line 91 – Can you define what laboratory-confirmed infection means, e.g., PCR test?

Line 92 – Can you expand on why certain participants continued to provide samples 4-10 months after infection? Were these convenience samples?

Line 133 – Please give more information on the antibodies used, e.g., vendor and catalog number.

Line 133/138 – first use of OD and RLU, please define.

Results

Line 119 – Please provide the catalog number for the peptide M agarose beads.

Line 149 – “Skimmed acellular milk was aliquoted and frozen at -80o C until testing. Undiluted milk samples obtained 4-6 weeks post-infection from 75 COVID-19-recovered donors, and 20 pre-pandemic milk samples obtained prior to December, 2019 were screened in our IgA ELISA against recombinant trimeric SARS-CoV-2 Spike.” This is redundant with the methods and can be omitted.

Line 151 – extra comma in “December, 2019”

Lines 171/172 – The samples highlighted are missing the “b” suffix present in the figures. Please clarify.

Line 200 – Should “of the period of follow-up” be “at follow-up”?.

Line 200 – Can the authors comment on the appropriateness of performing statistics comparing mean endpoint titers by pooling samples collected from 4-10 mo post-infection? Were other statistical tests incorporating the interval between collections considered?

Line 201 – Figure 3, panel A would be improved by making it more apparent which donors saw increases, decreases, and no change in IgA over time by coloring them by these factors. Similarly, Panel B would be improved by coloring donors by the groups highlighted in lines 206-208.

Line 204 – Even if not showing the data, please include the number of samples included in the subset. Compared to earlier figures, Fig. 4 could include these data as an additional panel. I would suggest showing these results or highlighting them in panel B (if colored as mentioned above they could be further distinguished with a “#” above the bars or below each joint ID).

Line 209 – “as with the larger durability cohort” – I thought these donors were part of this cohort, but were they not? Please clarify or rephrase.

Lines 220 – Were the milk samples tested the 4-6 week samples or the 4-10 month samples, please clarify.

Discussion

Line 266 – While the milk IgG response is very likely to be less robust than the milk IgA response, this was not demonstrated in the current manuscript, and has yet to be demonstrated with SARS-CoV-2. Consider omitting this statement on the robustness of IgG or provide a citation.

A Limitations section is missing. There is a lack of participant characteristics detailed in this current manuscript. As such it is difficult to determine if these results are generalizable to all lactating women or limited to specific demographics. Another limitation might be that IgG was not isolated and tested for the ability to neutralize SARS-CoV-2.

Figures

The figures would be improved by removing the full sentence descriptions within panels and instead only listing the most pertinent information when necessary, e.g., Fig. 2, panels A and B could be “Spike-specific secretory component” and “Spike-specific IgG”, respectively.

Fig. 1, panel B – the title over the plot indicates that prepandemic controls are included here, but this differs from the legend. Please clarify.

Fig. 1, panel C – Consider ordering the milk samples along the y-axis by endpoint titer (rather than ID). This would greatly aid in easily visualizing the amounts of samples with endpoint titers crossing each threshold.

Error bars should be defined.

Minor

The title could be improved by making it more concise. It is also somewhat circular to state “The Spike-specific IgA in milk elicited after SARS-Cov-2 infection is … strongly correlated with IgA binding”; not much would be lost by omitting some of the more declaratory adjectives.

The v in “Cov” in the title of the manuscript should be capitalized.

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Reviewer #1: No

Reviewer #2: No

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The IgA in milk induced by SARS-CoV-2 infection is comprised of mainly secretory antibody that is neutralizing and highly durable over time

PONE-D-21-10237R1

Dear Dr. Powell,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Etsuro Ito

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

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.

Reviewer #2: All comments have been addressed

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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.

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

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.

Reviewer #2: Yes

**********

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.

Reviewer #2: Yes

**********

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 #2: The authors have addressed all of my comments. The current version of the manuscript reads well and I have no further comments.

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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.

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 #2: No