Dear Ms. Gostic,

Thank you very much for submitting your manuscript "Childhood immune imprinting to influenza A shapes birth year-specific risk during seasonal H1N1 and H3N2 epidemics" (PPATHOGENS-D-19-01238) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

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Sabra L. Klein

Guest Editor

PLOS Pathogens

Andrew Pekosz

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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All reviewers commented on the importance of addressing early life exposure in immune imprinting to influenza A viruses in humans. While the reviewers found the question important a few major concerns were raised that must be addressed. First, concerns were raised about biases in surveillance data collection, which can differ by age (Reviewer 3). Also, how host-related factors, including sex, in addition to age, could impact early life imprinting (Reviewer 1) is not considered in the the analyses to ensure that the conclusions drawn are valid across individuals.

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The goal of this paper was to assess how childhood immune imprinting shapes seasonal influenza epidemiology. The authors use large epidemiological surveillance data to test whether this immune imprinting acts primarily by immune memory of a particular influenza subtype or via broader immunity that protects again various subtypes. The authors show that within subtype imprinting has the strongest impact on seasonal influenza risk and that antibody responses acquired later in life do not have the same impact on long-term immunity as early-life influenza responses. This is a well-written manuscript with important implications. My specific comments are below:

Reviewer #2: Overall this is a very insightful manuscript that describes the role of early life influenza exposures in protection against currently circulating seasonal influenza strains. This is a topic of great importance and the manuscript itself is well written with clearly presented data. The authors have published a previous paper examining how early life influenza exposures provide some degree of protection against potentially pandemic influenza strains. However, as humans are repeatedly exposed to seasonal influenza via both vaccination and infection, the protection afforded against circulating strains via imprinting could be vastly different. Indeed, the authors do find early life influenza exposures are able to imprint immunity and provide significant but only subtype-specific protection against future infections into adulthood and beyond. Further, a novel role for the NA protein in imprinting is described. Overall this paper represents an important contribution to the field and the data appear to support the conclusions being drawn.

Reviewer #3: This is a well-written manuscript by Gostic and colleagues examining imprinting, a critical question in the influenza field. The modeling is a real strength, but I have significant concerns about the data that is modeled.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Assessment of host factors

Although the authors investigate how age can impact imprinting responses, they do not provide the sex of the individuals in their study, nor do they account for sex in their analyses. Previous work has shown that sex can impact influenza responses in human and animal models, and it is important that in this type of epidemiological study, host factors, including sex, are included. Further, although I recognize it is difficult to access human data over the life course, it would be important to know more about the patients included in the analysis and if any patients had co-morbidities that may have influenced their responses both early on and later in life. With increasing age, susceptibility to infection increases; antibody responses and vaccine efficacy decrease; and sex hormones decrease. These immunological and endocrinological changes are important to account for when assessing how age and year of birth may impact responses to influenza.

Reviewer #2: None

Reviewer #3: 1. This manuscript relies completely on influenza surveillance data, however, no detail is provided on the surveillance system. In particular, typically state surveillance systems are designed in such a way that they target specific at-risk populations. Thus, the data is collected in an intentionally biased way and does not represent what is happening at a population level, but rather maximizes the cases identified. For instance, sentinel surveillance systems tend to have over-representation of pediatric offices to catch non-severe cases, outbreaks tend to be investigated (and reported on) in old age homes, and hospital surveillance may be more heavily done in specific adult hospitals. In addition, the design of the surveillance system may change over time. If severity patterns differ by virus, which is likely, and surveillance setting differs by age (which is also likely, but I am unfamiliar with surveillance in Arizona), then bias could be introduced into the models. The authors should report on the design of the surveillance system in the methods section including any changes over the study period and, most importantly, on how the design of the surveillance system may have impacted their results.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Childhood age range

The authors include “children” from 0 to 10 years of age. Have the authors thought about the fact that individuals within that large range may have varying degrees of imprinting and also exhibit vastly different immune responses (e.g., a 6-month old child will elicit different responses from a 10 year old). It would be helpful if the authors address this idea and suggest how they controlled for this in the current study.

Reviewer #2: While the discussion raises many important points, it is very long and would benefit from being compressed while still retaining the major points being made. Although outside of the scope of the current paper (and analyzed dataset), a future study performing similar analysis on a cohort on which serum/PBMCs could also be analyzed would provide significant insight on the immunologic mechanisms underlying the described findings.

Reviewer #3: 1. The authors should define imprinting more thoroughly in the introduction as non-influenza audiences may not be familiar with the term and in the influenza field imprinting has been used to describe several phenomena that may have different mechanistic bases (and indeed the authors themselves do this when using imprinting vs. within-subtype imprinting).

2. The authors state that HA group-level responses, which I assume includes the stem, are not thought to play a strong role in defense against familiar strains, however, a recent article by Ng et. al demonstrated that anti-HA stem antibodies are an independent correlate of protection.

3. Throughout the manuscript the authors refer to “infections” or “infecting” but they are looking at reported cases, not infections.

4. The authors suggest that imprinting may be a result of CD4 T-cells (as one possibility). Is there any evidence for this?

5. The authors use a case series. Did the population structure of Arizona change substantially over the time period? If so, they should adjust for the changes in the demographics.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Dear Ms. Gostic,

We are pleased to inform that your manuscript, "Childhood immune imprinting to influenza A shapes birth year-specific risk during seasonal H1N1 and H3N2 epidemics", has been editorially accepted for publication at PLOS Pathogens. 

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Best regards,

Sabra L. Klein

Guest Editor

PLOS Pathogens

Andrew Pekosz

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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The authors carefully considered the reviews and incorporated as many of the requested modifications as possible, given some limitations of the data set. This is a very important study that will make a significant impact on the field. There are no additional concerns or requests for modifications.

Reviewer Comments (if any, and for reference):