MAJOR ARTICLE

Age-Associated Cross-reactive AntibodyDependent Cellular Cytotoxicity Toward 2009
Pandemic Inﬂuenza A Virus Subtype H1N1
Sinthujan Jegaskanda,1 Karen L. Laurie,2 Thakshila H. Amarasena,1 Wendy R. Winnall,1 Marit Kramski,1 Robert De Rose,1
Ian G. Barr,2 Andrew G. Brooks,1 Patrick C. Reading,1,2 and Stephen J. Kent1
1

Background. During the 2009 pandemic of inﬂuenza A virus subtype H1N1 (A[H1N1]pdm09) infection, older
individuals were partially protected from severe disease. It is not known whether preexisting antibodies with effector
functions such as antibody-dependent cellular cytotoxicity (ADCC) contributed to the immunity observed.
Methods. We tested serum specimens obtained from 182 individuals aged 1–72 years that were collected either
immediately before or after the A(H1N1)pdm09 pandemic for ADCC antibodies to the A(H1N1)pdm09 hemagglutinin (HA) protein.
Results. A(H1N1)pdm09 HA-speciﬁc ADCC antibodies were detected in almost all individuals aged >45 years(28/31 subjects) before the 2009 A(H1N1) pandemic. Conversely, only approximately half of the individuals aged
1–14 years (11/31) and 15–45 years (17/31) had cross-reactive ADCC antibodies before the 2009 A(H1N1) pandemic. The A(H1N1)pdm09-speciﬁc ADCC antibodies were able to efﬁciently mediate the killing of inﬂuenza virus–
infected respiratory epithelial cells. Further, subjects >45 years of age had higher ADCC titers to a range of seasonal
H1N1 HA proteins, including from the 1918 virus, compared with younger individuals.
Conclusions. ADCC antibodies may have contributed to the protection exhibited in older individuals during
the 2009 A(H1N1) pandemic. This work has signiﬁcant implications for improved vaccination strategies for future
inﬂuenza pandemics.
Keywords.

ADCC; inﬂuenza; NK cells; swine-origin inﬂuenza virus.

The pandemic emergence of a swine-origin inﬂuenza A
virus subtype H1N1 during 2009 (A[H1N1]pdm09) resulted in signiﬁcant human morbidity worldwide [1–5].
Interestingly, individuals born before 1957 had lower rates
of infection and less severe disease than individuals
born after this time [2, 6–10], presumably from priorexposure to seasonal inﬂuenza A virus subtype H1N1
(sH1N1) [11–15]. The mechanism of natural protection

Received 9 February 2013; accepted 20 March 2013; electronically published 28
June 2013.
Presented in part: 42nd Annual Scientiﬁc Meeting of the Australian Society for
Immunology, 2–6 December 2012, Melbourne, Australia. Abstract 188.
Correspondence: Stephen Kent, MD, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia (skent@unimelb.
edu.au).
The Journal of Infectious Diseases 2013;208:1051–61© The Author 2013. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:journals.permissions@oup.com.
DOI: 10.1093/infdis/jit294

against A(H1N1)pdm09 infection has been largely attributed to cross-neutralizing antibodies and/or T-cell
responses induced following prior exposure to sH1N1
strains [11, 12, 16–22], but other antibody effector mechanisms may be involved and are unexplored.
Inﬂuenza virus hemagglutinin (HA)–speciﬁc antibodies have been mainly characterized for their neutralizing capacity. However, these antibodies have otherfunctions, including complement ﬁxation [23, 24],
phagocytosis [25], and antibody-dependent cellular cytotoxicity (ADCC) [26–30]. While neutralizing antibodies may target more variable regions found on theglobular head of the viral HA, ADCC antibodies may
target more conserved regions of the inﬂuenza virus HA
and other surface proteins [31, 32]. Recently, we developed new assays to measure ADCC antibodies against
multiple inﬂuenza virus strains [30]. We previously intensively studied ADCC responses in 10 healthy adults
to a broad range of inﬂuenza virus strains and detected

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Department of Microbiology and Immunology, University of Melbourne, Parkville; and 2WHO Collaborating Centre for Reference and Research on
Inﬂuenza, VIDRL, North Melbourne, Australia

ADCC antibodies to various inﬂuenza virus strains in theabsence of detectable neutralizing antibodies. However, our previous small human cohort study was limited by unclear exposure to speciﬁc inﬂuenza viruses. The emergence and rapid
spread of A(H1N1)pdm09 provided an opportunity to study
the cross-reactivity of ADCC antibodies against inﬂuenza virusthat were present before the pandemic. We hypothesized that
ADCC antibodies to A(H1N1)pdm09 were present in older individuals and may have potentially played a role in reducing
the severity of disease due to A(H1N1)pdm09. We studied
serum specimens obtained from 93 subjects before the 2009
H1N1 pandemic and from 89 separate unpaired subjects after
the 2009 H1N1 pandemic for ADCC to A(H1N1)pdm09.

Serum Samples and Donors

Serum samples collected from 93 individuals immediately
before the 2009 A(H1N1) pandemic winter season in Australia(21 November 2008–13 May 2009) and from 89 individuals immediately following the pandemic (3 August 2009–16 November 2009), were provide by the World Health Organization
Collaborating Centre for Reference and Research on Inﬂuenza
and approved by institutional review boards at each contributing institution. The Alfred Hospital ethics committee approved
the use of human donor blood (number 248/06). All subjects
provided written informed consent.
Inﬂuenza Virus Antigens and Hemagglutination Inhibition (HI)Assay

Mammalian-cell-expressed recombinant HA proteins were purchased from Sinobiologicals (Shanghai, China). The inﬂuenza
virus used was a reassortant virus propagated in embryonatedhen’s eggs expressing the HA and neuraminidase (NA) from
A/Auckland/01/2009 (A/California/07/2009-like 2009 pandemic
H1N1) with the internal genes from A/Puerto Rico/8/1934(H1N1). HI assays were performed as previously described [9].
Plate-Bound ADCC Natural Killer (NK) Cell Activation Assay

We recently described novel ADCC assays that measure antibodymediated NK cell activation [30], developed as modiﬁcations ofhuman immunodeﬁciency virus–speciﬁc ADCC assays [33, 34].
Brieﬂy, 96-well ELISA plate (Nunc, Rochester, NY) were coatedovernight at 4°C with puriﬁed inﬂuenza protein (400 ng/well) in
phosphate-buffered saline (PBS). Wells were washed with PBS
and incubated with heat-inactivated plasma/sera for 2 hours at 37°
C. Plates were washed with PBS, and 106 freshly isolated humanhealthy donor peripheral blood mononuclear cells (PBMCs) wereadded to each well. Brefeldin A (5 μg/mL, Sigma, St. Louis, MO),
monensin (5 μg/mL, BD Bioscience, San Jose, CA), and CD107aAPC-H7 (H4A3 clone, BD Bioscience, San Jose, CA) were added,
and plates were incubated for 5 hours at 37°C with 5% CO2. Cells
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Antibody-Dependent Cellular Viral Elimination Assay, Using a
Human Respiratory Epithelial Cell LineAs previously described [30], conﬂuent A549 cells (ATCC no.
CCL-185) were washed once with PBS and infected with 10
multiplicities of infection of A(H1N1)pdm09 for 1 hour at 37°C
in 10% CO2, washed with RF10, and incubated for 3 hours at
37°C in 5% CO2. Cells were removed from the monolayer by
use of trypsin versene, washed, resuspended in 200 μL, and incubated with 50 µL of phycoerythrin-conjugated anti–major
histocompatibility complex class I antibody (clone W6/32, eBioscience, San Diego, CA) for 20 minutes to facilitate identiﬁcation of target cells by a ﬂuorescence-activated cell sorter.
Following washing, 6 × 105 infected A549 cells were combinedwith 2 × 105 freshly isolated human PBMCs (E:T, 1:3) in a
96-well U-bottomed plates, with or without 10 μL of heatinactivated plasma and CD107a-APC-H7 (H4A3 clone, BD
Bioscience, San Jose, CA), and incubated for 6 hours at 37°C in
5% CO2. Following incubation, cells were stained with surface
antibodiesanti-humanCD3,CD14,andCD56asdescribedabove,
permeabilized, intracellularly stained with ﬂuorescein isothiocyanate–conjugated anti-inﬂuenzavirus nucleoprotein (431 clone,
Abcam, Cambridge, MA) antibody, ﬁxed, and acquired by ﬂow
cytometry. The reduction in infected cells was calculated as [(percentage of nucleoprotein-positive cells or total number of nucleoprotein-positive cells in the presence of plasma) – (percentage of
nucleoprotein-positive cells or total number of nucleoproteinpositive cells in the absence of plasma)]/[percentage of nucleoprotein-positive cells or total number of nucleoprotein-positive cells
in the absence of plasma] × 100.

Statistical Analyses

Statistical analyses used SPSS, version 18, software (IBM,Armonk, NY) and Prism GraphPad, version 6 (GraphPad Software, San Diego, CA). Data were analyzed by the t test (Figure 1B
and 1C, Figure 2A, and Figure 3), 1-way analysis of variance(ANOVA) together with Tukey post hoc tests (Figure 1D and1E), 2-way ANOVA together with Bonferroni comparisons(Figure 5A), and Pearson product-moment correlation coefﬁcient(Figure 4 and Figure 5B and 5C). Statistically signiﬁcant outliers
were determined by GraphPad Outlier calculator (α = 0.05).
Before 1-way or 2-way ANOVA, data were log10-transformed toequalize variances, according to the Levene test.

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METHODS

were then incubated with surface antibodies CD3-PerCP (clone
SP34–2), CD14-PE-Cy7 (clone M5E2), and CD56-APC (clone
B159, all from BD Bioscience) for 30 minutes at room temperaturein the dark. Cells were ﬁxed with 1% formaldehyde (Sigma,
St. Louis, MO) for 10 minutes and permeabilized using Permeabilizing Solution 2 for 10 minutes (BD Bioscience). Finally, cells
were incubated at room temperature for 1 hour with interferon γ(IFN-γ)–AlexaFluor700 (clone B27, BD Bioscience).

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Figure 1. Antibody-dependent cellular cytotoxicity (ADCC)–mediated natural killer (NK) cell activation to seasonal inﬂuenza A virus subtype H1N1
(sH1N1) and 2009 pandemic inﬂuenza A virus subtype H1N1 (A[H1N1]pdm09). A, Gating strategy of NK cell activation assay. Intracellular expression of interferon γ (IFN-γ) was determined in CD14–CD3–CD56+ lymphocytes. B–E, The NK cell expression of IFN-γ and CD107a in response to plate-bound sH1N1
HA (B and D; A/Brisbane/59/2007) or A(H1N1)pdm09 HA (C and E; A/California/04/2009) proteins in the presence of either sera collected before the 2009
A(H1N1) pandemic (Pre-2009) or after the 2009 H1N1 pandemic (Post-2009). A total of 93 Pre-2009 specimens and 89 Post-2009 specimens were evaluated. Comparisons were made using t tests. F and G, NK cell expression of IFN-γ in response to sH1N1 (F) or A(H1N1)pdm09 (G) hemagglutinin (HA) protein

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RESULTS
Subjects and Inﬂuenza Virus HI Antibodies

A(H1N1)pdm09-Speciﬁc ADCC Antibodies in Serum Obtained
From Individuals Before and After the 2009 A(H1N1) Pandemic

We ﬁrst assessed ADCC by using a plate-bound NK cell activation assay [30] (Figure 1A). Studying the entire cohort, wefound that ADCC-mediated NK cell activation (as measured byboth IFN-γ and CD107a expression) against A(H1N1)pdm09(A/California/04/2009) HA protein was signiﬁcantly higher insera collected after the 2009 A(H1N1) pandemic, comparedwith sera collected before the 2009 A(H1N1) pandemic (meanIFN-γ expression [±SEM] by NK cells, 1.59% ± 0.18% before
the 2009 pandemic vs 2.57% ± 0.20% after the 2009 pandemic;
P < .001, by the t test; Figure 1C). We observed no signiﬁcant
increase in antibody-mediated NK cell IFN-γ or CD107a expression against sH1N1 (A/Brisbane/59/07) HA protein
between samples taken before and those taken after the 2009 A(H1N1) pandemic (mean IFN-γ expression [±SEM] by NK
cells, 1.83% ± 0.18% before the 2009 pandemic vs 1.99% ±0.21% after the 2009 pandemic; P = .54, by the t test; Figure 1B).
These data conﬁrm that A(H1N1)pdm09 infection results inenhanced A(H1N1)pdm09-speciﬁc ADCC antibodies.
During the 2009 A(H1N1) pandemic, older individuals had areduced incidence of infection with A(H1N1)pdm09, suggestiveof a degree of protection against A(H1N1)pdm09 infection [2,Increase in A(H1N1)pdm09–Speciﬁc ADCC Titer Following the2009 A(H1N1) Pandemic

The percentage of NK cells expressing IFN-γ at a single serum
dilution is an effective means of comparing ADCC activity between different serum samples but may not accurately quantify
the level of ADCC antibodies. Therefore, serial dilutions ofeach sample were performed, and we determined the dilution
at which NK cell activation falls below a threshold of 3 times
the background level of activation. We used a randomly selected subset of 30 sera samples collected before the 2009 A(H1N1)
pandemic and 24 sera samples collected after the 2009 A(H1N1)
pandemic (from individuals aged 15–45 years and those aged
>45 years only). ADCC titers against A(H1N1)pdm09 HA weresigniﬁcantly higher in samples taken after the 2009 A(H1N1)
pandemic, compared with samples taken before the 2009 A(H1N1) pandemic (mean titer, 95.7 before the 2009 pandemic vs364.2 after the 2009 pandemic; Figure 2A). The increased ADCC
titers following the 2009 A(H1N1) pandemic suggests the hypothesis that preexisting ADCC antibodies may assist in clearing
pandemic inﬂuenza virus infection and reducing illness in these
individuals.

Figure 1 continued. in samples collected before the 2009 A(H1N1) pandemic and after the 2009 A(H1N1) pandemic from individuals aged 1–14, 15–45,
and >45 years. All samples were corrected for background on the basis of their response to wells containing sera but with no plate-bound antigen. The
number of specimens range from 23 (Post-2009; ages 1–14 years) to 46 (Post-2009; ages 15–45 years). Lines depict the mean of each group. Comparisonswere made using 1-way analysis of variance followed by the Tukey post hoc test on log10-transformed data.
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It is not clear whether antibody effector mechanisms, including
ADCC antibodies, contributed to reduced A(H1N1)pdm09associated disease observed during the 2009 A(H1N1) pandemic. To study this, serum and plasma samples were collectedfrom 93 individuals immediately before the occurrence of the2009 A(H1N1) pandemic winter season in Australia and from89 individuals immediately following the 2009 A(H1N1) pandemic season and analyzed for ADCC-based immunity to pandemic inﬂuenza virus. All samples were ﬁrst tested by HI assays
for reactivity toward seasonal (A/Brisbane/59/2007) or pandemic (A/California/7/2009) H1N1 viruses (Table 1). Samples
collected from individuals before the 2009 A(H1N1) pandemic
had undetectable HI titers against A/California/07/2009 (<10),and samples collected after the 2009 A(H1N1) pandemic wererecruited on the basis of positive HI titers against this strain (>40).
Because previous studies indicated that individuals <45 years ofage showed a higher incidence of A(H1N1)pdm09 infection and agreater frequency of hospitalization with severe inﬂuenza [2, 6, 7],
we prospectively divided the samples into groups on the basis ofage (ie, 1–14, 15–45, and >45 years; Table 1).

6–10]. To determine whether older subjects had higher levels of
ADCC antibodies before the 2009 A(H1N1) pandemic, we prospectively sorted our data into 3 distinct age groups (1–14, 15–45, and >45 years). For each age group, ADCC responses to
sH1N1 (A/Brisbane/59/07) HA protein were similar in samples
taken before and after the 2009 A(H1N1) pandemic (Figure 1F).
In contrast, ADCC-mediated NK cell activation against A(H1N1)pdm09 HA protein was signiﬁcantly increased for individuals aged 1–14 years (P < .001) and those aged 15–45 years(P < .002) following the 2009 A(H1N1) pandemic (Figure 1G).
However, similar levels of antibody-mediated NK cell activation
against A(H1N1)pdm09 HA protein was observed in samples
taken before and after the 2009 A(H1N1) pandemic from individuals >45 years of age (mean IFN-γ expression [±SEM] on
NK cells, 2.56% ± 0.31% before the 2009 pandemic vs 2.80% ±0.30% after the 2009 pandemic; P = .993; Figure 1G). In addition, samples collected before the 2009 A(H1N1) pandemic
from individuals >45 years of age showed higher levels of NK
cell activation, compared with samples taken before the 2009 A(H1N1) pandemic from individuals aged 1–14 years (P < .001)
and those aged 15–45 years (P = .008). Thus, before the 2009 A(H1N1) pandemic, individuals >45 years of age commonly possessed preexisting crossreactive ADCC antibodies against A(H1N1)pdm09.

ADCC Antibodies Eliminate A(H1N1)pdm09–Infected
Respiratory Epithelial Cells

Figure 2. Higher antibody-dependent cellular cytotoxicity (ADCC) titers
following 2009 pandemic inﬂuenza A virus subtype H1N1 (A[H1N1]pdm09)
infection and correlation between ADCC and hemagglutination inhibition(HI) titer. A, ADCC antibody end point titers to A(H1N1)pdm09 (A/California/04/2009) hemagglutinin (HA) were determined by measuring the percentage of natural killer (NK) cells expressing interferon γ (IFN-γ) at 2-folddilutions of subject sera (1:10–1:640). End point titers were presented asmean + standard error of the mean (n = 30 before the 2009 pandemic [Pre2009] and n = 24 for after the 2009 pandemic [Post-2009]). Groups werecompared by the t test. A response was deemed positive when the frequency of NK cells expressing IFN-γ was at least 3 times background(without HA protein but with subject sera). B, Comparison of the frequency
of NK cells expressing IFN-γ in response to plate-bound A(H1N1)pdm09
HA protein in the presence of 1:10 dilution of sera, compared with the end
point titers to A(H1N1)pdm09 HA, using the same sera (n = 54). C, Comparison of end point ADCC titer and HI titer to A(H1N1)pdm09 (A/California/
04/2009 and A/California/07/2009 respectively; n = 24). Dots representing
subjects with the same HI and ADCC titers have been adjusted (for HI
titer) to visualize individual data points.

We next evaluated the functional capacity of ADCC antibodies
in sera obtained after the 2009 A(H1N1) pandemic to clear A(H1N1)pdm09-infected airway epithelial cells. Figure 3A shows
an example of the frequency of inﬂuenza virus nucleoproteincontaining cells in the absence (58.9%) or presence of a sera
samples from either before the 2009 A(H1N1) pandemic(60.9%) or after the 2009 A(H1N1) pandemic (29.1%). We calculated a reduction in the number of inﬂuenza-virus-infected
respiratory cells, from 14 308 (no serum) to 4967 (after the2009 A(H1N1) pandemic), for an absolute reduction of 65.3%.
In the same well, we observed a marked increase in the expression of the degranulation marker CD107a on NK cells, from
2.8% in the absence of sera to 25.6% in the presence of sera, ina sample obtained after the 2009 A(H1N1) pandemic (Figure 3A).
Comparison of the reduction of the number of A(H1N1)pdm09infected respiratory cells in the presence of either sera obtained
before or after the 2009 A(H1N1) pandemic showed that there
was a signiﬁcant increase in the elimination of virus-infected
cells in sera collected after the 2009 A(H1N1) pandemic (meanabsolution reduction [±SEM] of virus-infected cells, 5.9% ± 3.0%
before the 2009 pandemic and 36.4% ± 5.8% after the 2009

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Previous inﬂuenza virus ADCC studies assumed that the
proportion of NK cells activated by ADCC antibodies corresponds with the end point titer of ADCC antibodies measured
in this assay, but direct evidence is lacking [30]. To determine
whether the level of NK cell activation correlates with the
ADCC end point titer, we compared the percentage of ADCCactivated NK cells at a 1:10 dilution to the ADCC end point
titer for a number of different serum samples. A clear positive
association was observed between NK cell activation at a 1:10
dilution and the end point ADCC titer (Figure 2B).
In vaccination and infection studies, a HI antibody titer of>40 is generally regarded as protective [35]. Recently, we demonstrated that the presence of cross-reactive ADCC antibodies
to inﬂuenza virus can be present in the absence of neutralizing
antibodies [30]. Interestingly, across this cohort there was no relationship between the ADCC titer to A(H1N1)pdm09 HA andthe HI titer against A(H1N1)pdm09 (Figure 2C). Eleven of 24
individuals recorded a HI titer of 40 but an ADCC titer of≥160. Thus, the HI titer does not reﬂect the level of HA-speciﬁc
ADCC antibodies, highlighting a potential limitation of solely
using HI titer to identify predictive correlates of protection. It isalso notable that HI titers to the sH1N1 A/Brisbane/59/07 werehigher in the samples obtained before the 2009 pandemic, but
ADCC levels to A/Brisbane/59/07 were similar before and after
the 2009 pandemic (Figure 1B), further emphasizing the poorcorrelation between HI titers and ADCC antibodies.

pandemic; P < .001, by the t test; Figure 3B). Concomitantly,
there was increased CD107a expression on NK cells exposed tosera obtained after the 2009 A(H1N1) pandemic, compared with
sera obtained before the 2009 A(H1N1) pandemic (Figure 3C).
Together, this shows that ADCC antibodies after the 2009 A(H1N1) pandemic can mediate the elimination of A(H1N1)
pdm09-infected respiratory cells.
ADCC to sH1N1 Correlate With ADCC to A(H1N1)pdm09 in Older
IndividualsProtection of older individuals from A(H1N1)pdm09 infection
has been attributed to previous infections with 1918-like1056 • JID 2013:208 (1 October) • Jegaskanda et al

sH1N1 [11, 12, 17]. We compared antibody-mediated NK cell
activation against HA proteins from 2 sH1N1 vaccine strains(A/New Caledonia/20/99 and A/Brisbane/59/07) to the antibody-mediated NK cell activation against A(H1N1)pdm09 HA(A/California/04/09) protein, using all 89 sera samples collected immediately before the 2009 A(H1N1) pandemic (all individuals aged 1–14, 15–45, and >45 years). We found weak or
no association between ADCC responses (NK cell activation)
to sH1N1 HA proteins and ADCC responses to A(H1N1)
pdm09 HA protein for individuals aged 1–14 years (P = .01 andr 2 = 0.20 for A/New Caledonia/20/99; P = .34 and r 2 = 0.34 for
Brisbane/59/07; Figure 4A and 4B) and those aged 15–45 years

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Figure 3. Antibody-dependent cellular cytotoxicity (ADCC)–mediated elimination of inﬂuenza virus–infected respiratory cells. Human airway epithelial
cell line (A549) was infected with inﬂuenza virus (A/Auckland/01/2009-PR8) at a multiplicity of infection of 10. After 4 hours incubation, infected or uninfected A549 cells were combined with healthy donor peripheral blood mononuclear cells at 1:3 (E:T ratio) in the presence or absence of a 1:10 dilution ofsera. Representative plots of A549 cells expressing inﬂuenza virus nucleoprotein (NP; top) and CD56+CD3– natural killer (NK) cells expressing CD107a(bottom), with or without sera collected from individuals before the 2009 A(H1N1) pandemic (Pre-2009) or after the 2009 A(H1N1) pandemic (Post-2009).
B and C, The absolute reduction in A549 cells (B; n = 30 for Pre-2009 and n = 24 for Post-2009) and natural killer (NK) cell CD107a expression (C; n = 30
for Pre-2009 and n = 23 for Post-2009) was measured in the presence of sera from individuals obtained before the 2009 A(H1N1) pandemic and after the2009 A(H1N1) pandemic. Data are presented as mean + standard error of the mean. Groups were compared by the t test.

(P = .002 and r 2 = 0.29 for A/New Caledonia/20/99; P = .10 andr 2 = 0.09 for A/Brisbane/59/07; Figure 4C and 4D). However,there was a strong correlation between ADCC responses against
sH1N1 HA proteins and ADCC responses against A(H1N1)
pdm09 HA proteins for individuals >45 years of age (P < .001

and r 2 = 0.52 for A/New Caledonia/20/99; P < .001 and r 2 = 0.53
for A/Brisbane/59/07; Figure 4E and 4F). This suggests that
previous sH1N1 infections in older subjects contributed tothe development of cross-reactive antibodies against H1N1 A(H1N1)pdm09.

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Figure 4. Correlation of antibody-dependent cellular cytotoxicity (ADCC) antibody responses to 2009 pandemic inﬂuenza A virus subtype H1N1 (A[H1N1]
pdm09) and seasonal H1N1 viruses (sH1N1). Natural killer (NK) cell activation (measured as expression of interferon γ [IFN-γ] by CD56+CD3– cells) against
A(H1N1)pdm09 (A/California/04/2009) hemagglutinin (HA) was compared to NK cell activation against sH1N1 HA from A/New Caledonia/20/99 in thepresence of serum samples collected before the 2009 A(H1N1) pandemic (Pre-2009) from individuals aged 1–14 years (A), 15–45 years (C), or >45 years(E). NK cell activation against A(H1N1)pdm09 (A/California/04/2009) HA was compared to NK cell activation against sH1N1 HA from A/Brisbane/59/07 in
the presence of serum samples collected before the 2009 pandemic from individuals aged 1–14 years (B), 15–45 years (D), and >45 years (F). The Pearson
product-moment correlation coefﬁcient was used to determine when signiﬁcant correlations occurred. The number of specimens ranged from 31 (Pre-2009;
ages 15–45 years) to 46 (after the 2009 pandemic; ages 15–45 years).

Cross-reactive ADCC Toward 1918-Like Inﬂuenza Viruses

We further hypothesized that infection with A(H1N1)pdm09
might also increase the immunity to related H1N1 inﬂuenza
viruses by inducing cross-reactive ADCC antibodies in older
subjects. We compared antibody-mediated NK cell activation
against HA proteins from both the 1918 H1N1 HA protein andthe 1934 H1N1 strain (A/Puerto Rico/8/1934) HA protein,using a subset of 9 sera taken from individuals aged 15–45
years and 15 sera taken from individuals aged >45 years immediately following the 2009 A(H1N1) pandemic. There washigher antibody-mediated NK cell activation in response to HAproteins from H1N1 viruses from 1918 (A/Brevig Mission/1/
1918) and 2009 (A/California/04/2009) in individuals aged >45
years, compared with those aged 15–45 years (2-way ANOVA
with Bonferroni comparisons, P < .05 for A/Brevig Mission/1/
1918 and P < .01 for A/California/04/2009; Figure 5A). However,1058 • JID 2013:208 (1 October) • Jegaskanda et al

there was no signiﬁcant difference in antibody-mediated NK cell
activation between the 2 age groups when comparing HA proteins from H1N1 (A/Puerto Rico/8/1934) virus and HA proteinsderived from H3N2 (A/Brisbane/10/2007), H4N6 (A/Swine/
Ontario/01911–1/99), H5N1 (A/Anhui/1/2005), and H7N7
(A/chicken/Netherlands/1/03) viruses (Figure 5A).
If indeed A(H1N1)pdm09 infection boosted ADCC antibodies to 1918-like H1N1 strains, a correlation might be predicted
between antibody-mediated NK cell activation to HA proteinsfrom A(H1N1)pdm09 and 1918 H1N1. We compared antibody-mediated NK cell activation in response to A(H1N1)
pdm09 HA protein to that against 1918 (A/Brevig Mission/1/
1918) HA protein in individuals aged 15–45 years and those
aged >45 years after the 2009 A(H1N1) pandemic. Interestingly, ADCC responses to 1918 HA protein correlated strongly
with ADCC responses to A(H1N1)pdm09 HA protein in

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Figure 5. Antibody-dependent cellular cytotoxicity (ADCC) responses to 2009 pandemic inﬂuenza A virus subtype H1N1 (A[H1N1]pdm09) hemagglutinin(HA) correlates with ADCC antibodies to 1918-like H1N1 virus HA. A, Comparison of the frequency of natural killer (NK) cells expressing interferon
γ (IFN-γ) in response to plate-bound 1918 H1 HA (A/Brevig Mission/1/1918), PR8 H1 HA (/Puerto Rico/8/1934), 2009-H1N1 HA (A/California/04/2009), H3
HA (A/Brisbane/10/2007), H4 HA (A/Swine/Ontario/01911–1/99), H5 HA (A/Anhui/1/2005), or H7 HA (A/chicken/Netherlands/1/03) in the presence of a
1:40 dilution of sera from individuals aged 15–45 years or >45 years. Log10-transformed data were analyzed by 2-way analysis of variance, followed by
Bonferroni comparisons. *P < .05; **P < .01; n = 7 for each group. NS, nonsigniﬁcant. B and C, Comparison of NK cell activation to 1918 H1 HA (A/Brevig
Mission/1/1918) and NK cell activation to A(H1N1)pdm09 HA(A/California/04/2009), both in the presence of 1:40 dilution of sera collected from individuals
aged 15–45 years (B) or >45 years (C) after the 2009 A(H1N1) pandemic, analyzed by Pearson product-moment correlation coefﬁcient (n = 54).

Table 1. Age-Stratiﬁed Summary of Serum Hemagglutination Inhibition (HI) Titers in the Study Cohort Before and After the 2009 Pandemic of Inﬂuenza A Virus Subtype H1N1 Infection
HI Titer, Geometric
Mean (95% CI)

Time, Age

Subjects, No.

Age, Years, Median (Range)

Seasonal(A/Brisbane/59/2007)

Pandemic(A/California/07/09)

Before 2009a
Overall93

23 (1–59)

31.5 (−10.6 to 73.7)

5.1 (4.9–5.3)

1–14 y15–45 y31
31

4 (1–14)23 (15–46)

163.6 (56.7–270.6)
26.2 (−5.2 to 57.5)

5.2 (4.8–5.7)
5.1 (4.8–5.4)>45 y

52 (46–59)

7.3 (.7–14.0)

589

22 (1–72)

8.0 (−1.3 to 17.2)

97.2 (46.2–148.2)

1–14 y23

6 (1–14)5

146.2 (30.3–262.0)

15–45 y
>45 y

36
30

21 (16–45)
63 (46–72)

8.3 (−13.0 to 29.6)
11.5 (−.8 to 23.8)

95.1 (−1.6 to 191.9)
72.9 (41.3–104.6)

Abbreviation: CI, confidence interval.
aSamples were collected between 21 November 2008 and 13 May 2009, before the pandemic.

bSamples were collected between 3 August 2009 and 16 November 2009, after the pandemic. Samples were selected on the basis of the presence of HI titers of>40 to A/California/07/09.

individuals aged 15–45 years and those aged >45 years(P < .001; r 2 = 0.64 and r 2 = 0.68, respectively; Figure 5B and
5C). This suggests that infection with A(H1N1)pdm09 signiﬁcantly boosts cross-reactive ADCC antibodies to 1918-likeviruses.
DISCUSSION
Novel pandemic inﬂuenza viruses emerge regularly, leading
to widespread mortality and morbidity. The recent 2009 A(H1N1) pandemic affected individuals <40 years of age more
severely than older individuals [2, 6–10]. This suggests that
there is some level of cross-protection against A(H1N1)pdm09
in older individuals that is not found in younger individuals.
Although studies have shown that neutralizing antibodies andT-cell responses are critical in cross-protection [12, 13, 17, 21, 36],
here we show that other antibody effector mechanisms, suchas ADCC, may play an important role. Sera from individuals>45 years of age commonly contained preexisting ADCC antibodies to A(H1N1)pdm09. These ADCC antibodies not only activated NK cells but also eliminated A(H1N1)pdm09-infected
respiratory cells. Indeed, levels of A(H1N1)pdm09-speciﬁc ADCC
antibodies were signiﬁcantly increased following the 2009 A(H1N1)
pandemic. The ability of ADCC antibodies to protect against
novel inﬂuenza viruses is an important area for future research.
Recent animal model studies found that seasonal inﬂuenza
virus infection provides some protection from challenge with A(H1N1)pdm09 and that nonneutralizing antibodies may be involved [11, 17, 37–39]. A higher titer of A(H1N1)pdm09-speciﬁc
binding antibodies is induced by infection with sH1N1 thatcirculated before 1950 [11]. sH1N1 infection induced partial
protection against subsequent challenge with A(H1N1)pdm09
among macaques in the absence of detectable HI antibodies [17].
Although T-cell immunity likely plays a role in the protection
observed, subsequent analysis of serum samples from thatmacaque study by our group found that ADCC antibodies induced by the initial sH1N1 infection were cross-reactive with A(H1N1)pdm09 (unpublished data). We found that ADCC responses to 2 distinct sH1N1 viruses correlated with A(H1N1)
pdm09-speciﬁc ADCC in older individuals. This suggests thatprior exposure to seasonal inﬂuenza virus infections in older individuals induces cross-reactive A(H1N1)pdm09-speciﬁc ADCC.
Future studies in animal models can now focus on passive transfer
of monoclonal nonneutralizing antibodies that mediate ADCC, todeﬁne a role for ADCC in protective immunity.
HA-speciﬁc neutralizing antibodies generally recognize
highly variable antigenic regions on the globular head of HA
and tend to be highly speciﬁc for a particular strain of inﬂuenza
virus. We speculate that ADCC antibodies against conserved
regions of inﬂuenza virus HA may provide partial crossprotection from a range of inﬂuenza virus strains. We found nocorrelation between HI titers and HA-speciﬁc ADCC antibodies; this was expected since HI assays assess the binding of theantibody Fab region to bind antigen, whereas the NK cell assay
measures binding of the antibody Fc region to CD16 on NK
cells. Antibodies frequently target regions outside the receptorbinding domain of HA, and elderly individuals have a greater
antibody diversity and higher afﬁnity for the HA1 region of A
(H1N1)pdm09 HA, compared with younger individuals [37, 38].
ADCC antibodies directed to non-HA inﬂuenza virus proteins

Ab-Dependent Cellular Cytotoxicity to A(H1N1)pdm09 • JID 2013:208 (1 October) • 1059

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After 2009b
OverallNotes
Acknowledgments. We thank Emma Job, Louise Carolan, Julie
McAuley, and Lorena Brown for support and supply of reagents.
Financial support. This work was supported by the Australian National Health and Medical Research Council (awards 628331 and 510488). The
Melbourne WHO Collaborating Centre for Reference and Research on Inﬂuenza is supported by the Australian Government Department of Healthand Aging.
Potential conﬂicts of interest. All authors: No reported conﬂicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conﬂicts of Interest. Conﬂicts that the editors consider relevant to thecontent of the manuscript have been disclosed.

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