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Universal Vaccine Based on Ectodomain of
Matrix Protein 2 of Influenza A: Fc
Receptors and Alveolar Macrophages
Mediate Protection
Karim El Bakkouri, Francis Descamps, Marina De Filette,
Anouk Smet, Els Festjens, Ashley Birkett, Nico Van
Rooijen, Sjef Verbeek, Walter Fiers and Xavier Saelens
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http://www.jimmunol.org/content/suppl/2010/12/17/jimmunol.090214
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J Immunol 2011; 186:1022-1031; Prepublished online 17
December 2010;
doi: 10.4049/jimmunol.0902147
http://www.jimmunol.org/content/186/2/1022
The Journal of Immunology
Universal Vaccine Based on Ectodomain of Matrix Protein 2
of Influenza A: Fc Receptors and Alveolar Macrophages
Mediate Protection
Karim El Bakkouri,*,† Francis Descamps,*,†,1 Marina De Filette,*,† Anouk Smet,*,†
Els Festjens,*,† Ashley Birkett,‡,2 Nico Van Rooijen,x Sjef Verbeek,{ Walter Fiers,*,†
and Xavier Saelens*,†
V
accination against infectious diseases is one of the most
effective medical interventions. Multiple mechanisms of
immune protection exist, and which mechanism is predominant in a given vaccination/pathogen system is often unclear
or controversial. Nevertheless, knowledge of the in vivo mechanism of action of vaccines could help to develop improved immune
protection strategies against diseases for which effective vaccines
are currently unavailable or inadequate. In addition, a better insight
in the effector mechanisms is crucial for defining reliable correlates
of protection for novel vaccines.
Influenza virus is responsible for seasonal influenza epidemics
and infrequent, unpredictable pandemics. It has been estimated
*Department for Molecular Biomedical Research, Flanders Institute of Biotechnology
(VIB), B-9052 Ghent, Belgium; †Department of Biomedical Molecular Biology, Ghent
University, B-9052 Ghent, Belgium; ‡Acambis, Inc., Cambridge, MA 02139; xDepartment of Molecular Cell Biology, Vrije Universiteit, 1081 BT Amsterdam, The Netherlands; and {Department of Human Genetics, Leiden University Medical Center, 2333
ZA Leiden, The Netherlands
1
Current address: Ablynx NV, Ghent, Belgium.
2
Current address: The PATH Malaria Vaccine Initiative, Bethesda, MD 20814.
Received for publication July 7, 2009. Accepted for publication November 8, 2010.
This work was supported by National Institutes of Health Grant 5R01AI055632-02,
Acambis, Inc., European Union Grant COOP-CT-2005-017749, and Industrieel Onderzoeksfonds Stepstone Project IOF08/STEP/001 from Ghent University.
Address correspondence and reprint requests to Dr. Xavier Saelens, Department for
Molecular Biomedical Research, Department for Molecular Biomedical Research,
Flanders Institute of Biotechnology (VIB) and Ghent University, Technologiepark
927, B-9052 Ghent (Zwijnaarde), Belgium. E-mail address: xavier.saelens@dmbr.
vib-ugent.be
The online version of this article contains supplemental material.
Abbreviations used in this article: ADCC, Ab-dependent cell-mediated cytotoxicity;
AM, alveolar macrophage; BAL, bronchoalveolar lavage; HA, hemagglutinin; HBc,
hepatitis B virus core; i.t., intratracheal; M2e, matrix protein 2 ectodomain; NA,
neuraminidase; SPF, specific-pathogen–free.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902147
that each year 5–10% of the world population becomes infected with
influenza, resulting in considerable public health and economic
burdens (1). Annual influenza vaccination is recommended for
specific population groups, such as the elderly and individuals
suffering from diabetes or chronic respiratory or cardiovascular
diseases (1, 2). Currently licensed influenza vaccines are tripartite
and primarily aimed to provide protection by inducing Abs directed
against influenza A and B virus hemagglutinin (HA) and possibly
neuraminidase (NA) derived from two specific influenza A strains
and one B strain. Hemagglutination-inhibiting Ab responses in serum are believed to correlate with protection, provided that the HA
present in the vaccine corresponds fairly closely to that of the influenza strain circulating in the field (3–5). However, the antigenic
properties of HA and NA from epidemic influenza viruses vary repeatedly over time, a process known as antigenic drift driven by
escape selection from the adaptive immune response in the population (6, 7). Therefore, the composition of human influenza vaccines has to be revised each year (once for the northern and once for
the southern hemisphere) and adapted according to the results of
global surveillance performed by World Health Organization influenza reference laboratories. Surveillance and serological characterization of circulating influenza strains, recently supported by
antigenic cartography, allows predicting with a reasonable success
rate the identity of the epidemic influenza strains that are likely to
circulate in a subsequent year, although vaccine mismatches still
occur occasionally (8, 9).
Vaccines that target conserved influenza virus proteins can
protect against multiple influenza A virus subtypes in animal models and, pending further successful clinical development, remain
promising complements or alternatives for the HA-based, strainspecific vaccines currently used (10). We previously described
a universal influenza A virus vaccine based on the conserved
ectodomain of matrix protein 2 (M2e) (11). The M2e sequence is
conserved across all known human influenza A viruses (12). The
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The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e
sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal
influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the
in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in
wild-type, FcRg2/2, FcgRI2/2, FcgRIII2/2, and (FcgRI, FcgRIII)2/2 mice, we report in this study that Fc receptors are essential
for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcgRIII for in vivo
immune protection but other anti-M2e IgG isotypes can rescue FcgRIII2/2 mice from a lethal challenge. Using a conditional cell
depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune
protection. Additionally, we show that adoptive transfer of wild-type AM into (FcgRI, FcgRIII)2/2 mice restores protection by
passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected
cells are essential for protection by anti-M2e IgG. The Journal of Immunology, 2011, 186: 1022–1031.
The Journal of Immunology
Materials and Methods
Mice
BALB/c mice were purchased from Harlan, BALB/c FcRg2/2 (encoded by
Fcer1g) mice were from Taconic Farms, and C57BL/6 mice were from
Janvier. The mice were housed in individually ventilated cages in a specific-pathogen–free (SPF) animal house. The following mice were bred in
an experimental animal facility of DMBR under non-SPF conditions and
genotyped by a PCR protocol using genomic DNA from individual animals: FcgRI2/2 (encoded by Fcgr1), FcgRIII2/2 (encoded by Fcgr3), and
(FcgRI, FcgRIII) 2/2 (all in a BALB/c genetic background). All mice were
housed in a temperature-controlled environment with 12-h light/dark cycles and received water and food ad libitum. For experiments in which
SPF- (wild-type and FcR g2/2 BALB/c) and non-SPF–kept strains were
compared, the SPF-housed mice were transferred 3 wk in advance to the
non-SPF environment. Eight-week-old female mice were used in all
experiments. All animal experiments were done under conditions specified
by law (European Directive and Belgian Royal Decree of November 14,
1993) and reviewed and approved by the Institutional Ethics Committee on
Experimental Animals.
Passive immunization and virus challenge
To prepare high-titer M2e-immune serum, BALB/c mice were immunized
with the vaccine construct 1818, an M2e-hepatitis B virus core (HBc) fusion
protein containing three tandem copies of M2e fused to HBc, truncated at
amino acid residue 150 (26, 27). Ten micrograms M2e-HBc vaccine was
formulated with Ribi adjuvant (MPL + TDM; Sigma-Aldrich, St. Louis,
MO) as previously described (11, 27), and mice were immunized three
times i.p. at 3-wk intervals. Two weeks after the last immunization, the
mice were killed by cervical dislocation, and blood was collected by
cardiac puncture. Serum was prepared by clotting the blood at 37˚C for 30
min followed by centrifugation. Anti-M2e immune ascites fluid was prepared by immunizing BALB/c mice i.p. with 1818 vaccine formulated with
IFA. Between the second and third immunization, a 180/TG sarcoma cell
suspension was injected i.p. to induce the formation of ascites fluid, which
was collected 12 d after injection of cells. The resulting ascites fluid
contained anti-M2e IgG1, IgG2a, IgG2b, and IgG3 subtype Abs as determined with an M2e-peptide ELISA (27). Immune sera or purified IgG
isotype fractions were injected i.p. (200 ml/mouse, unless stated otherwise)
in naive mice. After 24 h, the mice were anesthetized with a mixture of
xylazine (10 mg/g) and ketamine (100 mg/g) and challenged by intranasal
administration of 50 ml PBS containing four LD50 of mouse-adapted X47
(PR8 3 A/Victoria/3/75) or A/PR/8/34 influenza A virus, as indicated in
the figure legends. Mortality and morbidity (weight loss) were monitored
for 14 d after challenge.
IgG isotype separation
Total IgG from 30 ml anti-M2e immune ascites was bound on an MEP
Hypercel column (Pall, Zaventem, Belgium) equilibrated with 50 mM
NaH2PO4 (pH 8), using an Åkta explorer chromatography system (GE
Healthcare, Diegem, Belgium). Under these conditions, all IgGs present in
the ascites fluid were captured on the column, whereas most contaminating
proteins were in the flow-through fraction. After washing with water, IgGs
were eluted in 50 mM citric acid buffer (pH 3), and the eluted material was
immediately neutralized with Tris-HCl buffer (pH 8.8). IgGs were then
fractionated on an rProtein A-Sepharose FF column (GE Healthcare).
Following equilibration with 50 mM NaH2PO4 buffer (pH 8), IgGs eluted
from the MEP Hypercel column were loaded on the protein A column. IgG
isotypes were eluted from the protein A column using a pH step gradient
with buffers of pH 4.5, 4, and 3. At each pH step, elution was repeated
three times with one column volume. The column was then flushed with
one column volume of 50 mM NaH2PO4 buffer (pH 8) before elution at the
next pH step. This purification procedure resulted in a pure IgG1 fraction
eluting at pH 4.5, a mixture of IgG1 and IgG2a in the pH 4 eluate, and
a mixture of predominantly IgG2b and residual IgG1 and IgG2a in the pH
3 eluate. This profile was determined by using M2e peptide ELISA and
a set of peroxidase-conjugated Abs specific for mouse isotypes IgG1,
IgG2a, IgG2b, or IgG3 (Southern Biotechnology Associates, Birmingham,
AL). The specificity of these secondary Abs for the different mouse IgG
isotypes was confirmed by using three mouse mAbs directed against DNP
(2,4-DNP) of different subclasses (IgG1, IgG2a, and IgG2b). The Abs
were kindly provided by Dr. J.-P. Coutelier (Université Catholique de
Louvain, Louvain-la-Neuve, Belgium). ELISA plates were coated with 10
mg/ml DNP-albumin to capture these IgG mAbs, and the captured Abs
were subsequently used to evaluate the specificity of the subclass-specific
secondary Abs used in this study (Supplemental Fig. 2).
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first 9 aa residues are almost completely conserved between human and avian influenza A viruses, and 7 out of the 15 remaining
amino acid residues may differ between human and other influenza A viruses. However, only two possible amino acid residues
appear to be well tolerated at these positions in M2e. Mice vaccinated with M2e-carrier constructs and subsequently challenged
with homosubtypic or heterosubtypic influenza virus infection
have much reduced morbidity and decreased lung virus titers and
are protected from virus-induced death (11). M2e-specific Abs
are responsible for the protective immunity induced by M2e
vaccination. Indeed, protective immunity can be transferred
from vaccinated animals to naive recipients by serum (11, 13–16).
Moreover, anti-M2e mAbs protect wild-type and immunodeficient
scid recipient mice against influenza A virus infection (17–19).
Although a contribution by M2e-specific T cell responses to
protection cannot be ruled out, M2e-specific serum Abs are crucial
for immune protection. But how anti-M2e Abs protect against an
influenza A virus challenge is still unclear. M2 is sparsely present
on virions but abundantly on virus-infected cells (20, 21), and
influenza infectivity in tissue culture cannot be neutralized by
anti-M2e Abs. The monoclonal anti-M2e Ab14C2 (IgG1) has
been reported to inhibit plaque size growth of some influenza
strains in vitro (20). However, other strains, such as PR8 and
WSN, which were not susceptible to the 14C2 effect on plaque
growth, were equally suppressed in vivo in M2e-VLP–immunized
mice as in other strains (11). Because anti-M2e Abs do not neutralize influenza viruses, M2e-based vaccines are considered infection permissive.
Abs in immune serum can bind M2 on the surface of infected
cells, but the ensuing mechanism of protection by anti-M2e Abs
remains poorly understood. Contradictory findings have been reported regarding the involvement of Ab-dependent cell-mediated
cytotoxicity (ADCC) by NK and/or NKT cells (15, 16). In addition, protection of mice by passive transfer of M2e-specific human
IgG1 (homologous to murine IgG2a) mAb was dependent on
a functional FcgRIII and complement pathway, whereas a human
IgG4 (homologous to murine IgG1) anti-M2e Ab failed to protect
mice (19). In contrast, Jegerlehner et al. (15) excluded a role for
complement based on the observation that C3-deficient mice were
as protected as wild-type mice by passively transferred mouse
anti-M2e hyperimmune serum.
In general, mouse IgG2a and IgG2b Abs restrict viral infections
better than IgG1 Abs (22, 23). This is in line with the requirement
for Th1 cytokine responses, such as IFN-g for Ig class switching
and associated enhancement of cellular antiviral responses (22–
24). This is further confirmed by the observation that a subtype
switch from IgG1 anti-M2e monoclonal 14C2 to the switch variant anti-M2e IgG2a improves its protective efficacy in vivo (17,
25).
We report on the crucial role of Fc receptors in anti-M2e IgGmediated protection by using four different mutant BALB/c mouse
strains with the same genetic background: FcRg2/2, FcgRI2/2,
FcgRIII2/2, and (FcgRI, FcgRIII)2/2. In addition, we demonstrate that both murine anti-M2e IgG1 and IgG2a/2b isotypes can
protect mice from influenza A virus challenge. FcgRIII2/2 mice
were also protected by IgG2a Abs directed against M2e, indicating that, at least for these isotypes, an Fc receptor-mediated
mechanism did not involve NK cells, which, in mouse, exclusively
express FcgRIII. In contrast, anti-M2e IgG1 Abs failed to protect FcgRIII2/2 mice. Finally, by using an in vivo cell-depletion
method and by adoptive transfer of wild-type alveolar macrophages into (FcgRI, FcgRIII)2/2 animals, we identified a critical
role for alveolar macrophages in protection by anti-M2e Abs.
1023
1024
FcRS AND ALVEOLAR MACROPHAGES IN ANTI-M2E IMMUNE PROTECTION
Depletion of alveolar macrophages
Liposomes containing dichloromethylene diphosphonate (clodronate) or
PBS were prepared as described previously (28). For liposome administration, BALB/c or C57BL/6 mice were anesthetized by i.p. injection with ketamine/xylazine and placed in a supine position at a head-up angle of ∼30˚.
One hundred microliters PBS-liposomes or clodronate-liposomes were administered slowly intratracheally (i.t.) via the mouth using a gel loading tip
attached to a Gilson pipette. Depletion of alveolar macrophages (AM) was
determined in control experiments by bronchoalveolar lavage (BAL) 24 h
after liposome administration under anesthesia with an i.p. injection of
avertin (2.5% in low endotoxin PBS). A 23-gauge cannula was installed into
the trachea, and cells were collected by washing the airway lumen with 2 3
0.5 ml PBS. After cytocentrifugation, cells were stained on cytospin preparations with May-Grunwald-Giemsa (Sigma-Aldrich). Twenty-four hours
after PBS-liposome or clodronate-liposome administration, mice were injected i.p. with 400 ml anti-M2e–HBc hyperimmune or naive control mouse
serum. After 24 h, a blood sample was taken from each mouse to determine
the serum anti-M2e IgG titer by peptide ELISA, and mice were challenged
with 4 LD50 mouse-adapted X47 virus as described above.
Adoptive transfer of alveolar macrophages
FACS analysis of single lung cell suspension
Single lung cell suspensions from mice treated with clodronate- or PBSencapsulated liposomes were prepared and analyzed as follows. One day
after treatment, mice were sacrificed by cervical dislocation, and lungs were
removed aseptically, cut in small pieces, and incubated with collagenase D
(100 ml 2 mg/ml) in 500 ml PBS at 37˚C. After 45 min, the cell suspensions were filtered through a 70-mm cell strainer and treated with a 190mM ammonium chloride solution (pH 7.2) to lyse RBCs. The resulting
single-cell suspension was fixed with 2% formaldehyde, incubated with
rat anti-F4/80, a pan-macrophage–specific marker (clone C1:A3-1; MorphoSys AbD) and labeled with a secondary Alexa Red-conjugated goat
anti-rat Ab. Fluorescence of anti-F4/80–labeled cells was measured using
a BD Biosciences FACSCalibur flow cytometer and CellQuest software
(BD Immunocytometry Systems, San Jose, CA).
Cell-mediated cytotoxicity assay
Splenocytes were prepared from mice sacrificed on days 1, 2, and 4 after i.t.
treatment with clodronate- or PBS-encapsulated liposomes and assayed for
NK and macrophage cytotoxicity using Yac-1 and L929 cells as targets,
respectively. One million target cells were labeled with Calcein-AM (5 mM;
Invitrogen) for 30 min and transferred to a 96-well round-bottom plate at
10,000 target cells per well. Spleen cells were then added as effectors at
E:T ratios of 100, 50, 25, and 12.5. After 5 h of incubation at 37˚C, the
fluorescence at 485 nm in the cell supernatant was measured using a
fluorometer (CytoFluor, PerSeptive Biosystems, Framingham, MA). The
spontaneous release is defined as the fluorescence in the supernatant of
Calcein-AM–labeled target cells without effectors; maximum release is the
fluorescence in the supernatant of Calcein-AM–labeled target cells treated
with 1% Triton 3100. The percentage of killing was calculated as follows:
specific release (%) = 100 3 ([fluorescence, experimental release] 2
[fluorescence, spontaneous Release])/([fluorescence, maximal release] 2
[fluorescence, spontaneous release]). All tests were carried out in triplicate.
Statistics
Statistical significance of differences between survival rates was analyzed
by comparing Kaplan-Meier curves using the log-rank test and MedCalc
Results
Protection by anti-M2e IgG Abs requires FcRg
Previous studies have demonstrated a strong correlation between
the levels of anti-M2e IgG Abs in serum induced by M2e-based
vaccines and protection against a potentially lethal influenza A
virus challenge (11, 13, 16, 26). In addition, passive transfer of
anti-M2e mAbs or M2e immune serum can protect mice from
influenza A virus challenge (11, 18, 19). To better understand the
in vivo protective mechanism of M2e-specific humoral immunity,
we compared the protective efficacy provided by passive transfer
of anti-M2e immune serum into wild-type and FcRg2/2 BALB/c
mice (29). This FcRg subunit is required for Ab-mediated
phagocytosis by macrophages and for NK-dependent ADCC.
Twenty-four hours after passive transfer and before challenge,
anti-M2e IgG titers and Ab isotype distribution were similar in
wild-type and FcRg2/2 animals (Fig. 1A). As expected, wild-type
recipient mice were protected from challenge with 4 LD50 mouseadapted X47 virus and displayed only transient weight loss (Fig.
1B, 1C). By contrast, all but one FcRg2/2 mice (n = 10) died of
influenza infection despite the presence of mouse anti-M2e immune serum (Fig. 1B). This lack of protection was statistically
highly significant (p , 0.0001 by Kaplan-Meier survival analysis)
and was also reflected in the morbidity profile, with mutant mice
displaying faster weight loss during the infection (Fig. 1C). In
addition, on day six after challenge with 1 LD50 mouse-adapted
X47 virus, the lung virus titer was 40 times higher in FcRg2/2
than in wild-type mice (data not shown). To exclude the possibility
that FcRg2/2 mice are intrinsically more sensitive to influenza A
virus challenge, the lethality of different viral challenge doses was
compared in naive wild-type and mutant animals. As shown in
Fig. 1D (and in agreement with Ref. 30), both mouse strains were
equally susceptible to influenza A virus challenge. Because the
FcR g-chain is essential for assembly and intracellular signaling
of the activating receptors FcgRI, -III, and -IV, we conclude that
one or more of these three known receptor(s) associated with the
common g-chain is/are essential for anti-M2e IgG-mediated immune protection.
FcgRI and FcgRIII play a partially redundant role in
protection by M2e-specific Abs
To identify the FcgR(s) that mediate protection by M2e-specific
Abs, we performed passive transfer experiments in FcgRI2/2,
FcgRIII2/2, and (FcgRI, FcgRIII) 2/2 knockout mice, followed by
lethal influenza A virus challenge. Wild-type and FcRg2/2 mice
were included as controls. All animals had BALB/c genetic background and were of the same age (8 to 9 wk) and sex (female) to
eliminate or reduce as much as possible any influence of these
variables on the outcome (31). To allow optimal detection of protective efficacy, we used a relatively low-titer ascites preparation
containing anti-M2e IgG1, IgG2a, IgG2b, and IgG3 isotype Abs.
Twenty-four hours after passive transfer of this immune serum and
before challenge, blood samples were taken from individual mice
for analysis. An M2e-peptide ELISA revealed comparable IgG titers against M2e peptide in all five recipient mouse strains (n = 12
per group) on the day of challenge (Fig. 2A). In agreement with the
results reported above, 10 out of 12 wild-type mice were protected
following challenge with mouse-adapted X47 (H3N2) virus,
whereas only 1 out of 12 FcRg2/2 mice survived the challenge,
although the animals had similar anti-M2e Ab levels (Fig. 2B;
p , 0.005). In this experiment, (FcgRI, FcgRIII)2/2 mice were
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AM were isolated by BAL with HBSS-EDTA (HBSS with 0.5 mM EDTA) of
BALB/c and (FcgRI, FcgRIII)2/2 mice. Twenty mice were sacrificed as
donors for every group of 12 mice. A 23-gauge cannula was inserted into the
trachea, and cells were collected by washing the airway lumen with 3 3 0.5
ml HBSS-EDTA. The obtained BAL fluid was centrifuged, and cells were
washed twice with PBS, counted, and resuspended in PBS at a density of
107 cells/ml. For adoptive transfer, mice (BALB/c wild-type and [FcgRI,
FcgRIII]2/2) were anesthetized by i.p. injection of ketamine/xylazine and
placed in a supine position at a head-up angle of ∼30˚. One hundred microliters PBS containing 106 cells was administered slowly i.t. using a gel
loading tip attached to a Gilson pipette. Wild-type mice received AM from
(FcgRI, FcgRIII)2/2 mice and, reciprocally, (FcgRI, FcgRIII)2/2 mice received AM from wild-type mice. Twenty-four hours later, two times six mice
were injected i.p. with 400 ml anti–M2e-HBc hyperimmune serum and two
times six mice with 400 ml PBS. After 24 h, a blood sample was taken from
each mouse to determine the serum anti-M2e IgG titer by peptide ELISA,
and mice were challenged with 2 LD50 mouse-adapted X47 virus.
Statistical Software (MedCalc, Mariakerke, Belgium). A value of p , 0.05
was considered statistically significant.
The Journal of Immunology
1025
FIGURE 1. FcRg is essential for protection by anti-M2e Abs against
influenza A virus challenge. A, Anti-M2e IgG subtype distribution in serum of BALB/c mice (wild-type, n = 12) and FcRg2/2 mice (n = 10) 24 h
after i.p. injection of hyperimmune anti-M2e murine serum. Shown are
mean endpoint titers (6 SD) of the indicated IgG subtypes in the two
mouse strains, as determined by ELISA. Survival (B) and morbidity (C)
of wild-type and FcRg2/2 mice after passive transfer of anti-M2e serum
followed by challenge with 4 LD50 mouse-adapted X47 virus. The morbidity curve depicts the percentage mean body weight relative to the
weight on the day of challenge (day 0) 6 SD. D, Wild-type and FcRg2/2
mice are equally susceptible to influenza A virus challenge. Eight-weekold female mice (n = 5) from both strains were challenged with two different doses of mouse-adapted X47 virus: 1 3 1023 (corresponding to ∼4
LD50 for wild-type BALB/c mice) and 1.1 3 1024 dilution of virus.
significantly less protected than wild-type mice (p , 0.005) and
FcgRIII2/2 mice (p = 0.01). The kinetics of death after challenge in
these double-knockout mice was also significantly slower compared with FcRg2/2 mice (p , 0.05). However, 2 wk after challenge,
mortality was similar in the two strains. Protection from death in
FcgRIII2/2 mice (9 out of 12 mice survived) was similar to that in
wild-type animals (10 out of 12 mice survived). Only 50% of the
FcgRI2/2 mice, however, survived the challenge, but this survival
rate was not significantly different from that of the wild-type group
(p = 0.09) or the (FcgRI, FcgRIII)2/2 mice (p = 0.21). Similar
results were obtained in a second passive transfer experiment followed by challenge with mouse-adapted PR8 virus (H1N1) (Supplemental Fig. 1). From these results, we conclude that loss of only
FcgRI or only FcgRIII does not result in an appreciable drop in
protection, whereas knockout of both FcgRI and FcgRIII strongly
reduces protection by anti-M2e Abs against influenza virus
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FIGURE 2. FcgRI and FcgRIII play a partially redundant role in protection by anti-M2e IgG. A, Endpoint anti-M2e IgG1, -2a, -2b, and -3 titers
in pooled serum samples taken 24 h after passive transfer of murine antiM2e immune serum into wild-type, FcRg2/2, FcgRI2/2, FcgRIII2/2, and
(FcgRI, FcgRIII)2/2 mice (n = 12 per group, before challenge). Anti-M2e
titers were also determined in serum prepared from blood samples taken
from surviving mice 14 d after challenge (indicated as After challenge). B,
Survival of the different anti-M2e immune serum-recipient mouse strains
after challenge with 4 LD50 mouse-adapted X47 virus.
1026
FcRS AND ALVEOLAR MACROPHAGES IN ANTI-M2E IMMUNE PROTECTION
challenge. Presumably, loss of FcgRI or FcgRIII alone is compensated sufficiently by other FcgRs. The difference in survival between the double-knockout and FcRg-deficient mice was
statistically significant in both experiments (p , 0.05, X47 challenge; and p , 0.005, PR8 challenge), suggesting a possibly auxiliary role for FcgRIV, which preferentially binds IgG2a and
IgG2b Abs (32), in anti-M2e IgG-dependent immune protection.
Anti-M2e IgG1 protects wild-type but not FcgRIII2/2 mice
Protection by anti-M2e Abs depends on alveolar macrophages
FcgRIII is expressed on macrophages, neutrophils, eosinophils,
NK cells, and a subset of T cells (37). It has been shown that
phagocytosis by macrophages of IgG1-opsonized but not IgG2aand IgG2b-opsonized cells critically depends on FcgRIII (38). In
addition, macrophages are known to contribute to phagocytosis of
influenza virus-infected cells (39) and to clearance of influenza
virus by Abs raised by immunization with a conventional subunit
vaccine (30). Therefore, we assessed the contribution of AM
to protection by anti-M2e IgG Abs. AM were eliminated by i.t.
instillation in BALB/c mice of clodronate-loaded liposomes,
whereas control mice received PBS-loaded liposomes. After 24 h,
the mice were passively immunized with either control or anti-
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A role for NK cells but not for complement (15) and a role for both
complement and ADCC (19) in anti-M2e Ab-mediated protection
have been reported. Following passive transfer of anti-M2e Abs,
we observed only a small reduction in protection against influenza
A virus challenge in mice lacking FcgRIII (Fig. 2B). The affinity
of IgG for a particular Fc receptor differs according to the IgG
isotype (33, 34). Therefore, the protective polyclonal M2e immune serum was fractionated into individual IgG isotypes so that
were tested in parallel for their protective potential. IgG Abs were
first captured on an MEP-Hypercel column, thereby removing
most contaminating serum proteins, and then the IgG Abs were
retained on a protein A affinity column. By using a fine-tuned pH
elution protocol, we were able to isolate a pure IgG1 fraction and
a fraction containing only IgG1 and IgG2a (Supplemental Fig. 2).
Other elution fractions had more complex compositions and were
not used. Total M2e immune serum and both of the separated IgG
isotype fractions were first characterized by i.p. injection in wildtype mice. Care was taken to administer equivalent anti-M2e titers
of each given IgG isotype to all mice (Fig. 3A). One day after
passive transfer, mice were infected with 4 LD50 PR8 virus. The
total anti-M2e immune serum and the IgG1 fraction, as well as
the IgG1 and IgG2a fraction, protected BALB/c mice against an
otherwise lethal virus challenge dose (Fig. 3B).
Next, we compared the protection by total serum and by the IgG1
fraction in FcgRIII2/2 BALB/c mice. The Fc-dependent in vivo
activity of mouse IgG1 Abs is mediated almost exclusively by the
low affinity FcgRIII receptor (35, 36). Consistent with the results
mentioned above (Fig. 2B), FcgRIII2/2 mice were well protected by
total anti-M2e immune serum (Fig. 3C). By contrast, purified antiM2e IgG1 failed to protect FcgRIII2/2 mice (p , 0.005, KaplanMeyer survival analysis, compared with FcgRIII2/2 mice receiving
total serum). Protection from a lethal influenza infection was restored in the FcgRIII2/2 animals that received the pH 4 eluate
fraction containing anti-M2e IgG1 and IgG2a, indicating that
interactions between anti-M2e IgG2a Abs and possibly (an)other
Fc receptor(s) can compensate for the loss of IgG1–FcgRIII cooperation in these knockout mice (Fig. 3B). On the basis of these
results, we conclude that protection of mice against a lethal influenza A virus challenge by anti-M2e IgG1 Abs requires FcgRIII.
However, IgG2a Abs, which bind with high affinity to FcgRI and
FcgRIV, can confer full protection in the absence of FcgRIII.
FIGURE 3. Protection by anti-M2e IgG1 depends on functional FcgRIII. A,
Murine M2e hyperimmune serum (serum) was fractionated by stepwise elution
using buffers of pH 4.5 and 4.0, as detailed in Materials and Methods.
Wild type, FcgRI2/2, FcgRIII2/2, (FcgRI, FcgRIII)2/2, and FcRg2/2 mice
received M2e immune serum by i.p. injection (n = 12 for each group). In addition, wild-type, FcgRI2/2, and FcgRIII2/2 mice received equivalent antiM2e titers of IgG1 by i.p. administration of serum fractions eluted at pH 4.5
(pure IgG1) and pH 4 (IgG1 and IgG2a). Shown are the mean (6 SD) endpoint
titers of anti-M2e IgG1, -2a, and -2b from blood samples taken 24 h after passive
transfer. Survival of wild-type (B) and FcgRI2/2 mice (C) passively immunized
as described in A, following challenge with 4 LD50 mouse-adapted PR8 virus.
M2e immune serum and subsequently challenged with 4 LD50
mouse-adapted X47 virus. Serum anti-M2e IgG levels in mice
treated with PBS-liposomes or clodronate-liposomes were
The Journal of Immunology
1027
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FIGURE 4. Alveolar macrophages are required for protection by anti-M2e IgGs. A, BALB/c mice were treated i.t. with PBS-loaded or clodronate-loaded
liposomes (n = 12 per group). Twenty-four hours later, mice received murine anti-M2e immune serum by i.p injection. The left bar in the graph represents
the anti-M2e IgG titer in the immune serum that was used for i.p. injection. The two bars on the right show the mean M2e-specific IgG endpoint titers (6
SD) in serum prepared from blood samples taken 2 d after anti-M2e serum injection of PBS- or clodronate-treated animals. B, Top panel: cytospin of cells
in the BAL of mice treated with PBS-liposomes or clodronate-liposomes. Cells were stained with May-Grunwald-Giemsa. Insets: left panel, intact AM;
right panel, apoptotic alveolar macrophage. Bottom panel: F4/80 expression determined by flow cytometry of single lung-cell suspensions prepared from
mice treated i.t. with PBS-loaded or clodronate-loaded liposomes. The histograms are representative of six mice per group. C, Survival (top panel) and
morbidity (bottom panel) of BALB/c mice receiving PBS or anti-M2e murine serum (anti-M2e IgG), depleted or not depleted of AM by i.t. administration
of clodronate-loaded or PBS-loaded liposomes, and subsequently challenged with 4 LD50 mouse-adapted X47 virus. Mice were treated with liposomes on
1028
FcRS AND ALVEOLAR MACROPHAGES IN ANTI-M2E IMMUNE PROTECTION
Discussion
In this study, we demonstrate that Fc receptors are essential for antiM2e IgG-mediated immune protection against influenza A virus
challenge. To document the functional interaction between Fc
receptors and anti-M2e IgGs, we used passive transfer of anti-M2e
immune serum into wild-type mice and four knockout mouse
strains, FcRg2/2, FcgRI2/2, FcgRIII2/2, and (FcgRI, FcgRIII)2/2,
matched for strain, sex, and age. To identify the role of the respective IgG isotype subclasses, we preferred to use a fractionated
polyclonal M2e immune serum rather than isotype variants of one
particular mAb because the humoral immune response against
M2e vaccine is polyclonal (40). We identified an essential func-
tional interaction between anti-M2e IgG1 Abs and FcgRIII in the
protection in the mouse model, whereas IgG2a might operate via
FcgRI and/or FcgRIV. Our results also provide evidence for
a crucial role of AM in the mechanism of protection provided by
anti-M2e Abs against influenza A virus challenge.
Our finding of a critical role for FcgRs in protection by antiM2e IgGs against a potentially lethal influenza A virus challenge
indicates that ADCC and Ab-dependent cell-mediated phagocytosis are the main mechanisms involved. Protection by M2e-based
vaccines depends on Abs (11, 15, 16). Unlike anti-HA Abs, antiM2e Abs do not prevent influenza virus infection in vitro, and
reduction of plaque size growth in the presence of an anti-M2e
mAb has been documented for certain strains but not for others,
such as PR8 virus, a challenge virus that we also used in this study
(11, 14, 20). M2 protein is expressed at least as abundantly as NA
on the surface of infected cells, but much less M2 protein molecules are incorporated into the virion compared with HA or NA
(12, 20). Therefore, anti-M2e IgGs probably provide protection by
interacting with virus-infected cells rather than by interacting with
viral particles. Through its Fc region, anti-M2e IgG can than bind
to and cross-link FcgRs expressed on myeloid cells. Depending on
the balance between engagement of activating or inhibitory IgGFcgRs, anti-M2e immune complexes could trigger immune effector cell activation, resulting in killing and/or phagocytosis of
infected cells and thereby suppressing progeny virus formation
and spread (34). Mice have the activating Fc receptors FcgRI,
FcgRIII, and FcgRIV and the inhibitory Fc receptor FcgRII. Individual Fc receptors bind IgG subclass Abs with different affinities, and the ratio of activating to inhibitory Fc receptor binding is
thought to determine the biological efficacy of a given IgG subclass Ab (34). We found that elimination of the common g-chain,
which is essential for intracellular signaling of the three activating
Fc receptors, abrogated protection by anti-M2e IgG. The loss of
protection in the FcRg2/2 mice was nearly complete, and it is
doubtful that an additional protection mechanism not involving
FcRg contributes substantially. There was, however, a significant
difference in protection between FcRg2/2 and (FcgRI, FcgRIII)2/2
mice, suggesting an additional contribution to protection by an
FcgRIV-mediated mechanism; FcgRIV is the most recently discovered FcgR family member (32). We also demonstrated that
purified M2e-specific IgG1 failed to protect FcgRIII2/2 animals
from a lethal influenza challenge. This is in line with the reported
phenotype of this knockout mouse and with the very low affinity
of the Fc domain of IgG1 for FcgRI and FcgRIV (34, 35). It is of
interest to note that protection by Abs induced by immunization
with conventional HA- and NA-based vaccines also critically depends on Fc receptors, despite the well known fact that anti-HA
Abs can have virus-neutralizing activity in vitro (30). Neutralization is measured in vitro and implies that the Abs can inhibit
entry of virus into target cells. However, based on in vivo studies,
it is likely that neutralizing antiviral Abs also act in concert with
cellular effector functions—for example, by opsonizing virions,
which are subsequently eliminated by phagocytes or by Abdependent killing of infected cells that display viral membrane
proteins at the cell surface, as was proposed for anti-HIV surface
Ag-specific Abs (41). Our results indicate that infected cells are
day 23, received naive or anti-M2e immune serum on day 21, and were challenged on day 0. The morbidity curve shows the percentage mean body weight
relative to the weight on the day of challenge (day 0) 6 SD. Representative graphs of three independent experiments are shown. D, Intratracheal administration of clodronate does not affect killing activity by NK cells or by macrophages in the periphery. Mice were treated with PBS-loaded or
clodronate-loaded liposomes administered i.t., and 1 (d1), 2 (d2), or 4 (d4) days later, splenocytes were used at different E:T ratios in an in vitro cell-killing
assay using Yac-1 (NK-target) or L929 (macrophage target) cells as targets.
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comparable (Fig. 4A). The effective elimination of AM in the
clodronate groups was demonstrated by cytospin slide analysis of
bronchoalveolar cells and by the loss of cells expressing the F4/80
cell surface marker in single lung cell preparations as determined
by FACS analysis (Fig. 4B). All mice that received control serum,
as well as those receiving the anti-M2e immune serum and that
were treated with clodronate-loaded liposomes, died after challenge (Fig. 4C). By contrast, all mice with intact AM (PBS-loaded
liposome recipients) that were treated with anti-M2e IgG immune
serum survived the challenge (p , 0.001, Kaplan-Meyer survival
analysis, compared with the chlodronate-liposome treated group
that received M2e-immune serum) (Fig. 4C). A similar experiment performed in C57BL/6 mice confirmed that AM are critical
for the protection by anti-M2e IgG (Supplemental Fig. 3). Intratracheal treatment with clodronate-loaded liposomes did not affect
the cytocidal activity of NK cells and macrophages in the periphery, as demonstrated using a YAC and L929 cell killing assay,
respectively (Fig. 4D). Therefore, we conclude that anti-M2e IgG
Abs protect mice from influenza A virus challenge by a mechanism that requires AM.
We next reverted the impairment of protection by anti-M2e
IgG in FcgR-deficient mice by adoptive transfer of wild-type
AM in the lung compartment. (FcgRI, FcgRIII)2/2 mice were
only slightly better protected than FcRg2/2 mice against influenza
A virus challenge after passive transfer of anti-M2e IgG (Fig. 2).
Because (FcgRI, FcgRIII)2/2 mice were breeding much better
than FcRg2 /2 mice, we used this double-knockout strain as
recipients for wild-type AM. Reciprocally, wild-type mice received AM from (FcgRI, FcgRIII)2/2 donors. After 24 h, the mice
were passively immunized with either control or anti-M2e immune serum and challenged the following day with 2 LD50
mouse-adapted X47 virus (Fig. 5A). Anti-M2e IgG recipient mice
had the same anti-M2e IgG serum titer on the day of challenge
(Fig. 5B). Intratracheal instillation of wild-type AM from wildtype naive donor mice restored protection against X47 virus
challenge in the presence of anti-M2e IgG (Fig. 5C). Anti-M2e
IgG-treated wild-type mice that had received AM from (FcgRI,
FcgRIII)2/2 donors were also protected, whereas all PBS treated
mice died. From this experiment, we conclude that adoptively
transferred wild-type AM into the lungs of (FcgRI, FcgRIII)2/2
mice restore protection by anti-M2e IgG in these knockout mice,
indicating that Fc receptors on AM play a key role in immune
protection.
The Journal of Immunology
eliminated by anti-M2e IgG-mediated cellular cytotoxicity or
phagocytosis because these cells express M2 abundantly at their
surface early postinfection. This mechanism allows removal of
infected cells before progeny virus budding and spread and explains the consistent drop in lung virus titers that we and others
observed in independent studies using experimental vaccines eliciting anti-M2e Abs (11, 14–16, 18, 27, 42–44). Opsonization of
virions by anti-M2e IgG followed by virion clearance might also
contribute to in vivo immune protection but is probably less effective given the low abundance of M2 molecules compared with
HA and NA on influenza viral particles (20).
Which cells are responsible for elimination of M2-expressing
cells in the presence of anti-M2e Abs? Jegerlehner et al. (15)
proposed an important role for NK-mediated Ab-dependent cytotoxicity in acquired anti-M2e immunity. However, two recent
studies concluded that NK cells are not essential for protection by
anti-M2e IgG or following vaccination with M2 expression vectors (16, 43). All three studies made use of Ab administration
to eliminate NK and NKT cells, either anti-NK1.1 (43) or antiasialo-GM1 (15, 16), but used different mouse strains [C57BL/6
(15, 43) and BALB/c (16) mice] and different influenza virus
challenge strains. Also, mice were actively immunized with an
rM2e-HBc protein vaccine (15), with M2-DNA followed by
a boost with an rM2-expressing adenoviral vector (16), or passively with anti-M2e mAbs (43). Considering these differences in
experimental protocols, it remains unclear whether there is a role
for NK cells in anti-M2e protective immunity induced by vaccination. Our results provide no evidence for a contribution by NK
cells to protective immunity, because NK cells from FcgRIII2/2
mice do not exhibit ADCC (38), and yet these mice were protected
to the same extent as wild-type mice after passive transfer of antiM2e IgG (Fig. 2B). However, an accessory role of NKs (e.g.,
acting in concert with AM and anti-M2e IgG) remains possible
and will require additional studies.
We demonstrated that AM play a pivotal role in the protection
by anti-M2e IgGs. AM are resident in the lung and are believed to
be among the first immune cells that encounter microbes in the
respiratory tract. These cells control the induction of primary
cytotoxic T cell responses (45) and the level of antiviral lung
cytokines, such as TNF, IFN-a, and IFN-g during influenza virus
infection of naive mice (46). It is likely that these innate antiviral
functions were also affected by selectively depleting AM with
i.t. instilled clodronate-loaded liposomes. However, in our experimental system, elimination of AM altered neither the morbidity
nor the mortality profile of control mice following challenge (Fig.
4, Supplemental Fig. 3). It may be concluded that in combination
with pre-existing anti-M2e Abs and, as demonstrated in this study,
in a process requiring FcgRs, AM suppress influenza virus infection and spread by eliminating infected cells more effectively
and rapidly than in naive animals. It has been shown that macrophages can eliminate apoptotic, influenza virus-infected cells
in vivo by phagocytosis, a process that appears to be promoted
by apoptosis of the target cells (39). Infiltration and activation
of lymphocytes is rapidly initiated following influenza virus infection, and these processes contribute critically to controlling the
infection. This is illustrated, for example, by the defective clearance of pulmonary virus in chemokine receptor 2 knockout mice
(47). It is possible that scavenger cells that infiltrate the lungs soon
postinfection also act in concert with anti-M2e IgGs to control
and eliminate influenza virus. However, our results reveal that resident AM are the most critical effecter cells responsible for antiM2e immunoprotection. Anti-M2e IgG-dependent cytotoxicity
directed against infected cells by ADCC and/or Ab-dependent cellmediated phagocytosis could thus operate at a very early stage
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FIGURE 5. Wild-type AM can restore protection by anti-M2e IgG
in (FcgRI, FcgRIII)2/2 mice. A, Overview of the protocol for adoptive transfer of alveolar macrophage. Wild-type BALB/c mice (n = 12)
and (FcgRI, FcgRIII)2/2 mice (n = 12) received AM from (FcgRI,
FcgRIII)2/2 and wild-type BALB/c mice, respectively. Twenty-four
hours later, six mice from each of these two groups received anti-M2e
IgG and six mice PBS by i.p. injection. B, Anti-M2e serum IgG titers in
wild-type and (FcgRI, FcgRIII)2/2 mice prior to challenge. Serum was
prepared from blood samples taken 24 h after i.p. injection of anti-M2e
IgG in wild-type (n = 6) and (FcgRI, FcgRIII)2/2 (n = 6) mice. The antiM2e IgG1 and IgG2a titers of the pooled serum samples were determined
by M2e peptide ELISA. The bars for the anti-M2e serum represent the
anti-M2e IgG titer in the immune serum that was used for i.p. injection.
C, Survival (top panel) and morbidity (bottom panel) of mice treated
as in A after challenge with 2 LD50 mouse-adapted X47 virus. The
morbidity curve shows the percentage mean body weight relative to the
weight on the day of challenge (day 0) 6 SD.
1029
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FcRS AND ALVEOLAR MACROPHAGES IN ANTI-M2E IMMUNE PROTECTION
Acknowledgments
We thank Dr. Patricia Patricia Londoño-Arcila and Dr. Amin Bredan for
critical reading of the manuscript and Frederik Vervalle and Wouter Martens for excellent technical assistance. We also thank Dr. Jean-Paul Coutelier (Université Catholique de Louvain, Louvain-la-Neuve, Belgium) for
providing IgG1, IgG2a, and IgG2b mouse mAb directed against DNP.
Disclosures
The authors have no financial conflicts of interest.
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postinfection with influenza A virus, allowing maximal impact on
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Ab-immune complexes trigger the classical pathway of complement activation, resulting in cell lysis. Jegerlehner et al. (15),
examining survival after virus challenge of C32/2 mice, concluded that complement does not play an important role in protection by passively transferred murine anti-M2e immune serum,
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to control lung virus titers in challenged mice. Our results suggest
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which have normal complement, were not protected after X47 or
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be protected from the deleterious consequences of infection by
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similar mechanisms of immune protection in humans to establish
a true correlate of protection for this universal influenza A vaccine.
The Journal of Immunology
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