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Brief Reviews
The
Journal of
Immunology
Influenza-Specific Antibody-Dependent Cellular
Cytotoxicity: Toward a Universal Influenza Vaccine
Sinthujan Jegaskanda,* Patrick C. Reading,*,† and Stephen J. Kent*
I
nfluenza virus has a large human and economic toll,
with seasonal influenza resulting in ∼300,000–500,000
deaths/y worldwide and more than an estimated $26.8–
87.1 billion/y in healthcare costs in the United States alone
(1, 2). Yearly vaccination is recommended to reduce the
burden of influenza disease. The fraction of severe illness
prevented for the 2012–2013 season was estimated by the
U.S. Centers for Disease Control and Prevention as being
a low 17.3% (confidence interval: 16.2–18.0%), with an estimated half of the U.S. population receiving the influenza
vaccine (3). Trivalent influenza vaccine preparations require
precise selection of circulating strains (typically an H1N1,
H3N2, and type B influenza virus) based on surveillance
data and have reduced protection when strains are mismatched
(4). Further, seasonal vaccines provide little protection from
avian-origin viruses, such as H5N1 and H7N9 (5). With
growing demand for broader therapeutic agents (6) and the
rising number of viral isolates resistant to current antiviral
treatments (7, 8), a broadly protective universal vaccine would
be an important development toward curtailing the impact of
influenza virus on the human population.
*Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, Victoria 3010, Australia;
and †World Health Organization Collaborating Centre for Reference and Research on
Influenza, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria
3051, Australia
Received for publication February 19, 2014. Accepted for publication April 29, 2014.
This work was supported by Australian National Health and Medical Research Council
Awards 628331 and 1023294. The Melbourne World Health Organization Collaborating Centre for Reference and Research on Influenza is supported by the Australian
Government Department of Health.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400432
Influenza A viruses (IAVs) are enveloped RNA viruses belonging to the family Orthomyxoviridae. The genome comprises eight ssRNA segments that encode for 11 distinct
polypeptides, including 8 structural viral proteins and 3
nonstructural proteins (Fig. 1A). The entry and release of
influenza virus from host cells is mediated by the surface
glycoproteins hemagglutinin (HA) and neuraminidase (NA),
respectively. Following receptor-mediated endocytosis, the
reduced pH within endosomes triggers a conformational
change in HA to facilitate fusion of viral and host cell
membranes and release into the host cytosol. The transmembrane M2-ion channel protein is involved in acidification of the virion, which is essential for uncoating of the virus.
The internal components of the influenza virus consist of
matrix 1 protein (M1); the ribonucleoprotein complex, which
consists of the viral RNA segments; the polymerase complex
proteins polymerase basic 1 (PB1), polymerase basic 2, and
polymerase acid; and nucleoprotein (NP). Nonstructural
protein 1, nonstructural protein 2, and PB1 frame 2 are only
found within infected cells and are responsible for overcoming
host immune mechanisms (9) (Fig. 1A).
Many studies have focused on the induction of broadly
neutralizing Abs (Nabs) or cross-reactive CTLs to define immune correlates of protection from influenza virus infection.
CTLs targeting peptides from NP or M1 were shown to be
effective in protecting from severe influenza infection (10–13)
and correlated with the reduction of symptomatic influenza
disease in humans (13, 14). Further, CTLs recognizing a
broad range of influenza viruses are present in healthy human subjects (15, 16). Conversely, Nabs directed to HA or
NA can inhibit virus entry or prevent virus release from host
cells, respectively (17, 18). Influenza vaccine studies primarily
measure hemagglutination inhibition (HI) titers, such Abs
that mediate HI target epitopes surrounding the receptorbinding site of influenza HA (Fig. 1B) (19, 20). However,
Abs that mediate HI tend to be highly specific for the
infecting virus or vaccine strain and amenable to escape
through point mutations or glycosylation of the receptor
binding site of the globular head. In contrast, subsets of Nabs
exist that do not mediate HI and bind the conserved helix-A
Address correspondence and reprint requests to Prof. Stephen Kent, Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, The
University of Melbourne, Parkville, VIC 3010, Australia. E-mail address: skent@unimelb.
edu.au
Abbreviations used in this article: ADCC, Ab-dependent cellular cytotoxicity; HA,
hemagglutinin; HI, hemagglutination inhibition; IAV, influenza A virus; M1, matrix
protein 1; NA, neuraminidase; Nab, neutralizing Ab; NP, nucleoprotein; PB1, polymerase basic 1.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
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There is an urgent need for universal influenza vaccines
that can control emerging pandemic influenza virus
threats without the need to generate new vaccines for
each strain. Neutralizing Abs to the influenza virus hemagglutinin glycoprotein are effective at controlling influenza infection but generally target highly variable
regions. Abs that can mediate other functions, such as
killing influenza-infected cells and activating innate immune responses (termed “Ab-dependent cellular cytotoxicity [ADCC]-mediating Abs”), may assist in protective
immunity to influenza. ADCC-mediating Abs can target
more conserved regions of influenza virus proteins and
recognize a broader array of influenza strains. We review
recent research on influenza-specific ADCC Abs and their
potential role in improved influenza-vaccination strategies. The Journal of Immunology, 2014, 193: 469–475.
470
BRIEF REVIEWS: INFLUENZA-SPECIFIC ADCC
region sitting between the HA1 and HA2 regions (known as
anti-stem Abs) (21–23). The stem region is highly conserved
among influenza viruses, and anti-stem Abs tend to mediate
their neutralization activity by either blocking viral fusion
with, or preventing cleavage of, the HA protein (24, 25).
Vaccine regimens that target the HA stem region have given
promising results in mouse models but have yet to be translated into human clinical studies (26–28).
Abs induced toward influenza virus can mediate a number of
nonneutralizing functions, including complement-mediated
lysis (29, 30), phagocytosis (31), and Ab-dependent cellular
cytotoxicity (ADCC) (32–36). In some instances, these nonneutralizing Abs mediate distinct antiviral activity; however,
in other cases, these additional functions can increase the
potency of Nabs. In this review, we describe previous studies
on influenza-specific ADCC and new technical developments
in the study of influenza-specific ADCC that may aid in future vaccine development.
Influenza-specific ADCC: past and present
ADCC uses effector arms of both the innate and adaptive
immune responses to kill target cells (Fig. 2). ADCC is initiated by secreted IgG Abs (IgG1 or IgG3 subtypes in humans
and IgG2a or IgG2b subtypes in mice) binding to Ags on the
surface of target cells. Effector cells, such as NK cells (but also
neutrophils and monocytes), which in humans express the
activating FcgRIIIa (CD16) receptor (in mice the FcgRIV
receptor has the highest affinity for IgG2a), are then able to
bind to the Fc region of the surface-bound Ab (37, 38). Ligation of the CD16 receptor on the effector cell leads to
phosphorylation of the C-terminal ITAM to activate Ca2+dependent signaling pathway, resulting in the release of preformed granzyme B and perforin granules from endosomes
(39). Both granzyme B and perforin cause DNA fragmentation and apoptosis of the target cell. Additionally, ligation of
the CD16 receptors on effector cells can result in the secretion
of several antiviral cytokines and chemokines, including
IFN-g, and TNF, and b-chemokines (e.g., MIP-1a and MIP1b), which have important antiviral and immunopathological
properties (40, 41).
Early studies performed during the late 1970s and the early
1980s established a role for ADCC against influenza virus.
In 1977 and 1978, Greenberg et al. (32, 33) suggested
that lymphocytes in peripheral blood were capable of mediating cytotoxicity toward influenza virus–infected cells in the
presence of small quantities of Abs secreted from PBMCs.
PBMCs isolated from healthy volunteers were found to mediate robust cytotoxicity toward either HeLa cells or BHK-21
cells infected with H3N2 (A/Hong Kong/2/68) virus and
labeled with [51Cr]. Greenberg et al. (42) later confirmed that
the presence of HA-specific Abs secreted by PBMCs correlated
with the ability of lymphocytes to mediate cytotoxicity against
influenza virus–infected cells. This is consistent with our recent
studies showing that cross-reactive ADCC-mediating Abs against
a broad range of HA and NA proteins from IAV strains were
present in serum from healthy volunteers (36).
Greenberg et al. (32, 42) also showed that vaccination with
an inactivated influenza vaccine or infection with influenza
virus resulted in increased cytotoxicity from PBMCs within
7 d. Hashimoto et al. (34) performed follow-up studies
showing that ADCC-mediating Abs were found in serum
from children recently vaccinated with either inactivated or
live-attenuated vaccines or infected with influenza virus. They
suggested that the cytotoxicity was likely the result of NK cell
activity. Our recent studies show that infection of macaques
with a seasonal H1N1 virus induces cross-reactive ADCCmediating Abs toward a heterologous H1N1 virus. These
ADCC Abs declined to lower, but still detectable levels, by
30 d postinfection. However, the ADCC Abs expanded significantly within 7 d postinfection with a heterologous H1N1
virus, highlighting the infection-mediated induction of memory B cells capable of producing ADCC Abs (43).
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FIGURE 1. Influenza virus and surface HA structure. (A) Influenza virus showing internal and external protein structure. (B) Influenza HA trimer; globular
head (red) and stem region (green). Images were generated from the crystal structure of HA from A/Aichi/2/1968 (H3N2) virus (PDB code: 2HMG) using
PyMOL software.
The Journal of Immunology
471
Recent studies by our group showed that healthy humans
have broad cross-reactive ADCC-mediating Abs, likely as a
result of multiple prior influenza infections. We found that
young adults with no known prior exposure to (consistent with
a lack of HI Abs that recognized) a 1968 H3N2 virus had robust
ADCC-mediating Abs to this strain (36). In our studies, most
influenza virus–specific ADCC-mediating Abs induced via
vaccination or infection were shown to be broadly reactive to
strains within a given subtype (32, 34, 44–46). However, we
detected cross-reactive ADCC-mediating Abs toward avian H5N1
and H7N7 strains in some individuals in the absence of prior
exposure (36). Presumably, these cross-reactive ADCC-mediating
Abs target conserved regions of the influenza HA, including the
stem region. This is consistent with our recent studies showing
a reduction in titers of ADCC-mediating Abs when measuring responses against the HA1 protein compared with the whole
HA protein (S. Jegaskanda, K. Vandenberg, K.L. Laurie, L. Loh,
M. Kramski, W.R. Winnall, K. Kedzierska, S. Rockman, and S.J.
Kent. manuscript in preparation). Such ADCC-mediating Abs are
likely expanded following heterologous influenza infection (43,
47, 48).
The levels of cross-reactive ADCC-mediating Abs in healthy
humans make measuring the generation of new ADCC
responses difficult. Abs and NK cells capable of mediating
ADCC are detectable in cord blood samples, suggesting that
ADCC-mediating Abs are present at low levels in all individuals
throughout their lifetime (35). This may shape the newborn’s
immune response to influenza virus, with the broader ADCCmediating maternal Abs having a greater protective effect on
the newborn (49, 50). We speculate that, in addition to the
waning of Nabs, the loss of maternal ADCC-mediating Abs
following cessation of breastfeeding may lead to increased influenza infections in infants. More detailed studies on the induction of cross-reactive Abs by multiple infections and the
influence of ADCC-mediating Abs on the protection of infants
are required to make such conclusions.
In vivo protection from influenza infection by ADCC-mediating Abs
The characterization of protective Ab responses to influenza
virus in mouse models has typically focused on Nab function.
However, recent studies suggest that the nonneutralizing functions provided by the Fc region of the Ab are important for
the potency and the protective ability of HA-specific Abs
(22, 51). A study by Corti et al. (22) showed that the broadly
neutralizing human FI6 Ab attributes most of its in vivo activity to its FcR-binding properties. Mice administered 3 mg/
kg of the FcR mutant (termed FI6-LALA) FI6 Ab had a 60%
reduction in survival compared with FI6 Ab or FI6 complement mutant (termed FI6-KA) when challenged with a lethal
dose of PR8 virus. However, the transfer of human IgG1 Abs
into mice that express mouse FcR may be suboptimal. A recent study by DiLillo et al. (52) showed that administration of
4 mg/ml of a mouse IgG2a form of FI6 Ab (which can mediate ADCC in mice) protected mice from lethal challenge,
whereas 4 mg/ml of mouse IgG1 FI6 Ab form (which does
not mediate ADCC in mice) did not. The protection afforded
by mouse IgG2a FI6 Ab was abolished when administered to
Fcer1g2/2 mice (mice lacking the FcRg-chain). Further, the
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FIGURE 2. Schematic diagram of ADCC to influenza virus–infected cells. IgG Abs (IgG1/IgG3 in humans or IgG2a/IgG2b in mice) bind to viral Ags
expressed on the surface of influenza virus–infected cells (left panel). Effector cells, such as NK cells (also neutrophils or monocytes), bind to the Ab Fc-region
using their FcgRIII receptor (in mice FcgRIV, middle panel). Upon Ab ligation, cytotoxic granules are released, and antiviral cytokines are expressed (right panel).
This results in apoptosis of the infected cell and a reduction in viral replication.
472
The influence of influenza ADCC-mediating Abs on the 2009
pandemic
In April 2009, a novel swine-origin H1N1 influenza virus
entered the human population and caused the first pandemic of
the 21st century (55–58). In contrast to seasonal influenza,
which has the highest morbidity and mortality in the elderly,
it was younger individuals (,60 y of age) who were most
affected during the 2009 pandemic. Older individuals seemed
to demonstrate a level of protection against severe A(H1N1)
pdm09 infection (58, 59). According to Hancock et al. (59),
a proportion of individuals (39/115, 34%) born before 1950
(.59 y of age) had detectable cross-reactive Nab titers against
the A(H1N1)pdm09 virus. However, the presence of Nabs
in some individuals may only partially explain the reduced
incidence of severe (H1N1)pdm09 virus infection in individuals .60 y of age. We recently showed that of the individuals .45 y of age with undetectable HI titers to A(H1N1)
pdm09, nearly all (28/31, 90%) had ADCC-mediating Abs to
the A(H1N1)pdm09 virus prior to the 2009 pandemic (44).
Numerous animal studies showed that infection with seasonal
H1N1 viruses, particularly with H1N1 viruses circulating
pre-1960, can provide a level of protection from severe A
(H1N1)pdm09 virus infection (21, 60–63). In studies in
which Nabs against A(H1N1)pdm09 could not be detected,
the levels of nonneutralizing binding Abs correlated closely
with protection from severe A(H1N1)pdm09 infection (12, 61).
Indeed, our studies showed that previous infection of macaques
with seasonal H1N1 virus induced cross-reactive ADCCmediating Abs toward A(H1N1)pdm09 (43). The expansion of
cross-reactive ADCC-mediating Abs targeting the stem region
of the influenza virus HA fits well with epidemiological studies.
Future passive-transfer studies and isolation of mAbs from donor
samples prior to the 2009 pandemic are required to determine
the targets and protective ability of polyclonal ADCC-mediating
Abs induced by seasonal H1N1 infection.
ADCC-mediated activity toward internal proteins of influenza virus
Vaccination strategies that target internal proteins of influenza
virus, namely NP and M1, have shown promise in animal models.
Amino acid sequences of NP and M1 are highly conserved across
different IAV subtypes and strains (i.e., ∼90% sequence identity),
making these proteins attractive vaccine targets. Until recently,
only T cell–mediated immunity toward internal proteins
was considered plausible for effective heterologous protection
from influenza virus infection. However, protein- or vector-based
vaccine constructs containing NP can induce nonneutralizing
Abs (64–67). Passive transfer of nonneutralizing polyclonal Abs
or mAbs toward NP was associated with protection from lethal
influenza challenge in mice (64, 68), although the mechanism of
Ab-mediated protection remains unclear. Of interest, studies
showed that intracellular NP Ag is transiently expressed on the
surface of influenza virus–infected cells (69, 70) and, therefore,
could represent a target for ADCC. Bodewes et al. (71) investigated the in vitro Fc-mediated effector functions of anti-NP
Abs, demonstrating that a human anti-NP Ab did not mediate
neutralization or complement-dependent cytotoxicity or improve
presentation by opsonization. Studies on HIV ADCC by our
group showed that ADCC can be mediated to peptides derived
from internal HIV proteins, including Pol and Vpu (72, 73).
Our limited studies in macaques suggest that anti-NP Abs mediate ADCC activity, because anti-NP Abs could activate macaque NK cells in vitro (46). Further studies are required to
determine whether virus-infected cells expressing NP can be
killed by ADCC, as well as the ability of anti-NP ADCCmediating Abs to mediate protection in vivo. The targeting of
conserved regions of NP or other internal proteins (M1, PB1)
for the induction of ADCC-mediating Abs may be a very useful
strategy for vaccine design (74, 75).
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human IgG1 form of the FI6 Ab was directly shown to mediate ADCC toward influenza virus–infected target cells. This
confirms the importance of ADCC for the protective activity
of the FI6 broadly Nab in mice.
DiLillo et al. (52) provide further support for the in vivo use
of influenza-specific ADCC, showing that, for HA stem Nabs,
the subclass and FcR function are important for protection
from lethal influenza infection. Mice administered the mouse
IgG2a subclass of the anti-stem 6F12 broadly Nab showed
reduced weight loss and increased survival following challenge
with a lethal dose of PR8 virus compared with those administered the mouse 6F12 IgG1 subclass (does not mediate
ADCC) or a 6F12 FcgR-binding–deficient Ab (DA265). The
protection provided by the IgG2a 6F12 Nab in mice was
FcgR dependent, because administration of IgG2 6F12 to
either Fcg-chain–deficient mice (Fcer1g2/2 mice; which lack
FcgRI, FcgRII, FcgRIII, and FcgRIV) or FcRa-null mice
(which lack FcgRI, FcgRIIb, FcgRIII, and FcgRIV) resulted
in reduced survival following lethal challenge with PR8. The
ability of stem Abs to mediate ADCC was shown by the activation of donor NK cells in the presence of human IgG1
6F12 and A549 cells infected with PR8 virus. In addition,
DiLillo et al. (52) showed that the anti-stalk Abs 2G02, 2B06,
and 1F02 protected mice from A(H1N1)pdm09 virus challenge in a FcR-dependent manner. Not surprisingly, FcR binding was critical for the potent protection provided by anti-stem
Abs because the mutant form of 6F12, deficient in FcR binding
(DA265), afforded protection only at concentrations .16 mg/
kg, whereas 4 mg/kg of unmodified IgG2a 6F12 provided
complete protection from lethal PR8 challenge. Indeed, these
findings may explain the ability of anti-stem Abs CR9114 and
CR8043 to protect mice from lethal challenge with a type B
virus or H7N7 virus, respectively, although these Abs did not
mediate in vitro neutralization of these viruses (21, 53).
However, the ability of Abs CR9114 and CR8043 to mediate
ADCC toward these strains needs to be assessed.
The investigation of stem Abs’ versus globular head Abs’ ability
to mediate ADCC is particularly interesting. DiLillo et al. (52)
suggested that only anti-stem Abs can bind in the correct confirmation to ligate FcRs. In their study, the one strain-specific
anti-HA head Ab (PY102) investigated was inefficient at mediating FcgR binding and ADCC activity; however, more Abs
to the globular head of HA need to be tested before clear conclusions can be drawn. Further, a polyclonal anti-HA ADCC
response may provide greater ligation of CD16, as seen for HIV
ADCC (54). Clearly, a larger subset of anti-HA head Abs needs to
be tested to determine their overall potential to mediate ADCC.
Our study and other studies characterized the role of NK cells in
mediating ADCC against influenza virus–infected cells (34–37).
However, other cells at the site of influenza infection, including
neutrophils and alveolar macrophages, express CD16 receptors
and could mediate ADCC activity. The relative importance of
different cell subtypes as effectors of ADCC activity is unclear.
BRIEF REVIEWS: INFLUENZA-SPECIFIC ADCC
The Journal of Immunology
Developing in vitro assays to measure influenza-specific
ADCC-mediating Abs
A possible road to universal influenza vaccines
Recent studies established a role for ADCC-mediating Abs
in protection from influenza virus infection. The importance
of ADCC function of Abs is becoming increasingly apparent
in other fields, with recent studies suggesting that the HIV
vaccine–mediated protection observed during the Thai
RV144 HIV study was due, in part, to ADCC-mediating Abs
(78). We speculate that the induction of ADCC-mediating
Abs to the conserved HA stem region may result in a partially
successful universal influenza vaccine. However, a stemspecific Ab response may be difficult to generate in all individuals, and the synthesis of large quantities of correctly folded recombinant HA stem without the immunogenic
globular head region will be technically challenging. Despite
this, we know that the potency of HA stem Abs is greatly
diminished if they do not have ADCC function. The
screening of distinct stem-specific Abs that bind, but do not
mediate neutralization, is possible with current in vitro
ADCC assays. Our current data suggest that most healthy
donors have persistent low-level cross-reactive ADCCmediating Abs. These cross-reactive Abs are found in individuals in the absence of detectable neutralization and suggest
that they are a distinct subset. The presence of these low-level
Abs implies that a pool of cross-reactive memory B cells may
exist that could be expanded with the appropriate vaccine
strategy. Future studies should aim to isolate and characterize
these B cells and Abs to determine whether they are different
from classical stem Abs.
Conclusions
Newly developed assays and the current interest in ADCCmediated responses to influenza virus are important for the
diversification of influenza immunity studies. The use of these
assays in other laboratories worldwide will drive a shift in perspective away from “neutralizing-only” Abs to Abs with a range
of other effector functions that also contribute to effective immunity. It is clear that ADCC-mediating Abs are found in most
individuals, and such Abs are cross-reactive against a broad
range of influenza subtypes. This represents a unique turning
point in our field and provides a possible avenue to assist in the
development of a universal influenza vaccine.
Disclosures
The authors have no financial conflicts of interest.
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The development of flow cytometry–based ADCC assays has
been important in the assessment and understanding of the
role of ADCC responses to influenza virus in humans. Recent
studies by our group (36, 43) and other investigators (52, 76)
described assays that measure NK cell activation following
incubation of donor PBMCs with either (mAbs or polyclonal
IgG) Ab-coated infected target cells or Ab–Ag complexes on
coated plates. In our studies, we coated recombinant influenza
HA proteins onto a 96-well plate, incubated them with serum
or purified Ig, and added PBMCs from healthy donors. We
then measured the activation of healthy donor NK cells by
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