Middle-aged individuals may be in a perpetual state of H3N2 influenza virus susceptibility

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Penn Medicine researchers have found that middle-aged individuals – those born in the late 1960s and the 1970s – may be in a perpetual state of H3N2 influenza virus susceptibility because their antibodies bind to H3N2 viruses but fail to prevent infections, according to a new study led by Scott Hensley, Ph.D., an associate professor of Microbiology at the Perelman School of Medicine at the University of Pennsylvania.

The paper was published today in Nature Communications.

“We found that different aged individuals have different H3N2 flu virus antibody specificities,” Hensley said. “Our studies show that early childhood infections can leave lifelong immunological imprints that affect how individuals respond to antigenically distinct viral strains later in life.”

Most humans are infected with influenza viruses by three to four years of age, and these initial childhood infections can elicit strong, long lasting memory immune responses.

H3N2 influenza viruses began circulating in humans in 1968 and have evolved substantially over the past 51 years.

Therefore, an individual’s birth year largely predicts which specific type of H3N2 virus they first encountered in childhood.

Researchers completed a serological survey—a blood test that measures antibody levels—using serum samples collected in the summer months prior to the 2017-2018 season from 140 children (ages one to 17) and 212 adults (ages 18 to 90).

They first measured the differences in antibody reactivity to various strains of H3N2, and then measured for neutralizing and non-neutralizing antibodies.

Neutralizing antibodies can prevent viral infections, whereas non-neutralizing antibodies can only help after an infection takes place.

Samples from children aged three to ten years old had the highest levels of neutralizing antibodies against contemporary H3N2 viruses, while most middle-aged samples had antibodies that could bind to these viruses but these antibodies could not prevent viral infections.

Hensley said his team’s findings are consistent with a concept known as “original antigenic sin” (OAS), originally proposed by Tom Francis, Jr. in 1960.

“Most individuals born in the late 1960s and 1970s were immunologically imprinted with H3N2 viruses that are very different compared to contemporary H3N2 viruses.

Upon infection with recent H3N2 viruses, these individuals tend to produce antibodies against regions that are conserved with older H3N2 strains and these types of antibodies typically do not prevent viral infections.”

According to the research team, it is possible that the presence of high levels of non-neutralizing antibodies in middle-aged adults has contributed to the continued persistence of H3N2 viruses in the human population.

Their findings might also relate to the unusual age distribution of H3N2 infections during the 2017-2018 season, in which H3N2 activity in middle-aged and older adults peaked earlier compared to children and young adults.

The researchers say that it will be important to continually complete large serological surveys in different aged individuals, including donors from populations with different vaccination rates. A better understanding of immunity within the population and within individuals will likely lead to improved models that are better able to predict the evolutionary trajectories of different influenza virus strains.

“Large serological studies can shed light on why the effectiveness of flu vaccines varies in individuals with different immune histories, while also identifying barriers that need to be overcome in order to design better vaccines that are able to elicit protective responses in all age groups,” said Sigrid Gouma, Ph.D., a postdoctoral researcher of Microbiology and first author on the paper.


Childhood influenza exposures leave an immunological imprint, which has reverberating, lifelong impacts on immune memory. Foundational work on original antigenic sin [1] and antigenic seniority [2] shows that individuals maintain the highest antibody titers against influenza strains encountered in childhood.

But how these serological patterns map to functional immune protection, and shape birth year-specific risk during outbreaks, remains an active area of inquiry. One open question is the breadth of cross-protection provided by immune memory imprinted in childhood.

We define immune imprinting as a lifelong bias in immune memory of, and protection against, the strains encountered in childhood. Such biases most likely become entrenched as subsequent exposures back-boost existing memory responses, rather than stimulating de novo responses [3].

By providing particularly robust protection against certain antigenic subtypes, or clades, imprinting can provide immunological benefits, but perhaps at the cost of equally strong protection against variants encountered later in life. For example, every modern influenza pandemic has spared certain birth cohorts, presumably due to cross-protective memory primed in childhood [4–10].

Recently, we showed that imprinting also protects against novel, emerging avian influenza viruses of the same phylogenetic group of hemagglutinin (HA) as the first childhood exposure [9,11]. Imprinting may additionally shape birth year-specific risk from seasonal influenza [12–14], but the importance of broadly protective immunity in this context is still being evaluated [15–17].

Until recently, narrow cross-protective immunity specific to variants of a single HA subtype has been considered the primary mode of defense against seasonal influenza.

Lymphocyte memory of variable epitopes on the HA head (i.e. sites at which hemagglutinin antigens of different subtypes show limited homology) drives this narrow, within-subtype protection, which is the main mechanism of protection from the inactivated influenza vaccine.

But a growing body of evidence shows protection may also be driven by memory of other influenza antigens (e.g. neuraminidase, NA) [18–20], or by immune response to conserved epitopes, many of which are found on the HA stalk [11,15,21–23].

Antibodies that target conserved HA epitopes can provide broad protection across multiple HA subtypes in the same phylogenetic group [21,23,24], where HA group 1 contains hemagglutinin subtypes H1 and H2, while group 2 contains H3. Only three HA subtypes have circulated seasonally in humans since 1918, H1, H2 and H3; H1 and H2 belong to phylogenetic group 1, while H3 is in group 2 [11,22,25].

Current thinking stipulates that within a single host, imprinting probably induces multiple levels of bias in immune memory, to both conserved (broadly protective) and variable (narrowly protective) sites on various influenza antigens.

The functional role of any single layer of imprinted immune memory depends on both immunodominance hierarchies and epidemic context. Here, we examine which layers of imprinted memory impact risk from seasonal influenza.

Different breadths of immunity are expected to act differently on influenza epidemiology. Within-subtype immunity to HA is known to shape seasonal influenza’s epidemiology and evolution [26], but due to rapid decay in the face of antigenic drift, it would not be expected to shape cohort-specific imprinting protection across an entire human lifetime [27,28].

Conversely, broad, HA group-level immune memory arises when lymphocytes target conserved HA epitopes. Responses to these conserved epitopes should be more stable over time, and can play a strong role in defense against unfamiliar influenza strains (e.g. novel, avian or pandemic subtypes [11,21,23,24,29,30], but are not traditionally though to act strongly against familiar, seasonal influenza subtypes.

However, responses to the conserved HA stem have recently been identified as an independent correlate of protection against seasonal influenza [15], and might play a particularly strong role against drifted seasonal strains whose variable HA epitopes have become unrecognizable.

Thus, childhood immune imprinting may determine which birth cohorts are primed for effective defense against seasonal strains with conserved HA epitopes characteristic of group 1 or group 2, or with variable HA epitopes characteristic of a particular subtype (H1, H2, etc.). A similar line of reasoning may apply to immunity against NA, although much less attention has been paid to this antigen.

Since 1977, two distinct subtypes of influenza A, H1N1 and H3N2, have circulated seasonally in humans, with striking but poorly understood differences in their age-specific impact [9,12–14,31]. These differences could be associated with childhood imprinting: older cohorts were almost certainly exposed to H1N1 in childhood (since it was the only subtype circulating in humans from 1918–1957), and now seem to be preferentially protected against modern seasonal H1N1 variants [9,12–14].

Likewise, younger adults have the highest probabilities of childhood imprinting to H3N2 (Fig 1), which is consistent with relatively low numbers of clinically attended H3N2 cases in these cohorts. Alternatively, differences in the evolutionary dynamics of H1N1 and H3N2 could explain the observed age profiles.

Subtype H3N2 exhibits slightly faster drift in its antigenic phenotype than H1N1, and as a result, H3N2 may be better able to escape pre-existing immunity in immunologically experienced adults, whereas H1N1 may be relatively restricted to causing disease in immunologically inexperienced children [32].

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Fig 1
Model and expectations under different imprinting hypotheses.
(A) Reconstructed, birth year-specific probabilities of imprinting (representative example specific to cases observed in 2015). Throughout the manuscript, group 1 HA subtypes are represented in blue and group 2 subtypes in red. (B) Expected imprinting protection against H1N1 or H3N2 under the three tested models. (C) Cartoon of expected age distribution of any influenza case, before controlling for subtype-specific imprinting. The shape of this curve is purely hypothetical, but each of our tested models combined demographic age distribution with a fitted, age-specific risk step function to generate similar, data-driven curves. (D-F) Fraction of each birth year unprotected by their childhood imprinting (from A) determines the shape of birth year-specific risk. (G-I) A linear combination of demography plus age-specific risk (as in C), and birth year-specific risk (as in D-F) give the expected age distribution of H1N1 or H3N2 cases under each model.

We analyzed a large surveillance data set of relatively severe, clinically attended influenza cases to test whether cohort effects from childhood imprinting primarily act against variable epitopes, only providing narrow cross-protection against closely related HA or NA variants of the same subtype, or against more conserved epitopes, providing broad cross-protection across HA subtypes in the same phylogenetic group (Fig 1A and 1B).

We fitted a suite of models to data using maximum likelihood and compared models using AIC. In a separate analysis, we considered the hypothesis that differences in evolutionary rate of H1N1 and H3N2, rather than imprinting effects, shape differences in age distribution. Our results have implications for long-term projections of seasonal influenza risk in elderly cohorts [13], who suffer the heaviest burdens of influenza-related morbidity and mortality, and whose imprinting status will shift through time as cohorts born during different inter-pandemic eras grow older.

Discussion
We analyzed a large epidemiological surveillance dataset and found that seasonal influenza subtypes H1N1 and H3N2 cause different age distributions of relatively severe, clinically attended cases, confirming previously reported patterns [12–14].

We analyzed several possible drivers of these differences systematically, and found the greatest support for imprinting protection against seasonal influenza viruses of the same NA or HA subtype as the first influenza strain encountered in childhood [12,13].

The data did not support strong effects from broader HA group-level imprinting, as recently detected for novel zoonotic or pandemic viruses [9,11], or from differences in rates of antigenic evolution [32].

Our results suggest individuals retain a lifelong bias in immune memory, and that this imprint is not erased even after decades of exposure to or vaccination against dissimilar influenza subtypes.

External evidence corroborates the idea that birth year, rather than age, drives subtype-specific differences in seasonal influenza risk. When H3N2 first emerged in 1968, it caused little or no excess mortality in the elderly, who had putatively been exposed, as children or young adults, to an H3 virus that had circulated in the late 1800s [7,9].

Meanwhile, H1N1-imprinted cohorts (those ~10–50 years old at the time) experienced considerable excess mortality in the 1968 pandemic [7]. Now, fifty years later, the same H1N1-imprinted cohorts continue to experience excess H3N2 morbidity and mortality as older adults [12–14,31] (Fig 2).

In model comparison, the data supported childhood imprinting to NA at the subtype level. Although NA is not as intensively studied as HA, these results emphasize the increasingly recognized importance of both antigens as drivers of protection against seasonal influenza [18–20].

Realistically, some combination of effects from both HA and NA subtype-level imprinting probably shapes seasonal influenza risk; both models of imprinting produced similar fits to data, and far outperformed other models in terms of AIC (Fig 3).

Unfortunately, due to the limited diversity of seasonal influenza subtypes that have circulated in humans over the past century, collinearities between even the relatively simple models tested here prevented us from testing more complicated models of combined effects from imprinting to multiple antigens.

Deeper insights into the respective roles of HA and NA will most likely need to come from focused immunological cohort studies, in which individual histories of influenza infection are recorded and can be studied alongside changes in serology, PBMCs, and/or the B cell repertoire [35].

Alternatively, the development of immunological biomarkers for diagnosis of imprinting status in individual patients could substantially increase the power of epidemiological inference.

We did not detect a clear relationship between annual antigenic advance and epidemic age distribution, although small sample sizes may have limited our statistical power. We did detect a weak trend, consistent with the idea that influenza cases are more restricted to immunologically inexperienced children in seasons of low antigenic advance, as previously proposed [32].

But the data did not reveal a clear relationship between antigenic advance and the fraction of cases occurring in adult age groups, where epidemiological data reveal distinct subtype-specific differences in impact.

Perhaps antigenic advance shapes how cases are distributed between children and adults, but has small or inconsistent impacts within the adult population. We speculate that clearer relationships between antigenic advance and epidemic age distribution might emerge if methods to estimate antigenic distance were able to incorporate effects such as immune history [42], glycosylation [42,43], and immunity to antigens other than HA [19,20,44].

The exact immunological drivers of imprinting protection against seasonal influenza remain unclear, but our results provide some new clues. Traditionally, within-subtype cross-protection is thought to decay quickly with antigenic drift.

Strains that circulated more than 14 years apart rarely show measurable cross-protective titers by the hemagglutination inhibition (HI) assay [40].

The short timescale of immune memory to variable HA head epitopes stands in contrast to patterns observed in our study and others [12–14], where within-subtype immune memory imprinted in childhood appears to persist for an entire human lifetime, remaining evident even in the oldest cohorts.

We speculate that within-subtype imprinting protection may involve epitopes that are more conserved, and stable over time, than those typically measured in HI assays.

These inform most existing estimates of antigenic distance, but disproportionately measure antibodies to variable, immunodominant epitopes on the HA head [15,24].

Across a lifetime of exposures to diverse H1N1 and H3N2 variants, repeated back-boosting of antibodies to intermediately conserved sites on HA or NA (i.e. sites conserved within but not across HA and NA subtypes), could explain the longevity of subtype-level imprinting protection. This is consistent with recent evidence that the immune repertoire shifts to focus on more conserved influenza epitopes as we age [27,28].

Another possibility is that memory B cell clones developed during the first childhood influenza infection may later adapt via somatic hypermutation to follow antigenic targets as they drift over time. However, this would be inconsistent with new evidence suggesting memory B cells are relatively fixed in phenotype, and have little potential for ongoing affinity maturation [45,46], or that somatic hypermutation decreases with age [27].

Finally, the role of CD4+ T cells in imprinting is unclear, but T cell memory and T cell help to B cells within germinal centers both play at least some role in the development of the immune repertoire [47].

Signals of imprinting protection are anomalously strong in the current cohort of elderly adults, as reflected by higher estimates of imprinting protection to H1N1 than H3N2. The oldest subjects in our data, born slightly after 1918, and would not have encountered an influenza virus of any subtype other than H1N1 until roughly age 30.

Repeated early-life exposures to diverse H1N1 variants may have reinforced and expanded the breadth of H1N1-specific immune memory [5,48]. But this strong H1N1 protection seems to come at a cost; even after decades of seasonal H3N2 exposure, and vaccination, older cohorts have evidently failed to develop equally strong protection against H3N2.

HA group 1 antigens (e.g. H1) appear to induce narrower immune responses, and less cross-group protection than structurally distinct HA group 2 antigens (e.g. H3) [25].

Perhaps elderly cohorts imprinted to group 1 antigens have been trapped in narrower responses that offer exceptional protection against strains similar to that of first exposure but relatively poor adaptability to other subtypes.

We speculate that imprinting protection, which currently limits the number of severe, clinically-attended H1N1 cases in the elderly, also limits the mortality impact of H1N1 viruses. Although pre- and post- 2009 H1N1 lineages have caused slightly different profiles of age-specific mortality [13], neither H1N1 lineage causes nearly as many deaths as H3N2 in high-risk elderly cohorts [13,31,49].

On the one hand, if strong subtype-specific biases from imprinting remain in future cohorts of elderly adults, our results would corroborate the idea that mortality from H1N1 may increase as protection in the elderly shifts from H1N1 toward other subtypes [9,13].

On the other hand, given that cohorts born after 1968 have had much more varied early life exposures to both H1N1 and H3N2, these cohorts may show a greater ability to act as immunological generalists as they become elderly, capable of effective defense against multiple subtypes.

Our study has several limitations. Relatively severe, clinically attended cases are much more likely to be detected, confirmed to subtype, and included in our data than mild cases. Thus, while our results show a clear relationship between subtype-level imprinting and risk of relatively severe, clinically attended influenza, the relationship between imprinting and mild or asymptomatic cases could not be determined from available data.

Given the limited number of variables recorded in the data, we could not model explicitly the impact of individual risk factors such as the presence of comorbidities, patient sex, or vaccination status. All these factors are known to shape immunity and influenza risk [50], and all may cause individual imprinting outcomes to vary from the average, population trends measured by our study.

Understanding how these patient-level covariates modulate imprinting and other aspects of immunity is the next frontier in this line of research. For now, working within the constraints of the available data, we designed the age-specific risk component of the model to capture empirically the combined effects of several risk factors that could not be modeled individually.

Additionally, we analyzed the relative count of H1N1 to H3N2 cases within each single year of birth, not absolute incidence, to control for minor age-specific biases in sampling, which are almost inevitably present in any large surveillance data set.

Another limitation was the low number of confirmed cases available in the pre-2009 era. Large, detailed data sets collected continuously over decades provide the greatest power to separate the effects of age from birth year. We emphatically echo earlier calls [51] for more systematic sharing of single year-of-age influenza surveillance data, standardization of sampling effort, and reporting of age-specific denominators, which could substantially boost the scientific community’s ability to link influenza’s genetic and antigenic properties with epidemiological outcomes. Additionally, collection and reporting of covariates such as sex, vaccination status and the presence of comorbidities in surveillance data would help us understand how patient-level variables modulate imprinting, and immunity in general [52,53].

Altogether, this analysis confirms that the epidemiological burden of H1N1 and H3N2 is shaped by cohort-specific differences in childhood imprinting [9,12,13,16,54], and that this imprinting acts at the HA or NA subtype level against seasonal influenza [16,17].

The lack of support for broader, HA group-level imprinting effects emphasizes the consequences of immunodominance of influenza’s most variable epitopes, and the difficulty of deploying broadly protective memory B cell responses against familiar, seasonal strains. Overall, these findings advance our understanding of how antigenic seniority shapes cohort-specific risk during epidemics.

The fact that elderly cohorts show relatively weak immune protection against H3N2, even after living through decades of seasonal exposure to or vaccination against H3N2, suggests that antibody responses acquired in adulthood do not provide the same strength or durability of immune protection as responses primed in childhood.

Immunological experiments that consider multiple viral exposures, and cohort studies in which individual histories of influenza infection are tracked from birth, promise to illuminate how B cell and T cell memory develop across a series of early life exposures. In particular, these studies may provide clearer insights than epidemiological data into which influenza antigens, epitopes and immune effectors play the greatest role in immune imprinting, and how quickly subtype-specific biases become entrenched across the first or the first few exposures.

reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6922319/


More information: Sigrid Gouma et al, Middle-aged individuals may be in a perpetual state of H3N2 influenza virus susceptibility, Nature Communications (2020). DOI: 10.1038/s41467-020-18465-x

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