MF59 and Pam3CSK4 Boost Adaptive

Responses to Influenza Subunit Vaccine

This information is current as
of September 19, 2013.
through an IFN Type I-Independent
Mechanism of Action
Elena Caproni, Elaine Tritto, Mario Cortese, Alessandro
Muzzi, Flaviana Mosca, Elisabetta Monaci, Barbara
Baudner, Anja Seubert and Ennio De Gregorio
J Immunol 2012; 188:3088-3098; Prepublished online 20
February 2012;

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doi: 10.4049/jimmunol.1101764
http://www.jimmunol.org/content/188/7/3088

Supplementary http://www.jimmunol.org/content/suppl/2012/02/21/jimmunol.110176
Material 4.DC1.html
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 The Journal of Immunology

MF59 and Pam3CSK4 Boost Adaptive Responses to Influenza

Subunit Vaccine through an IFN Type I-Independent
Mechanism of Action

Elena Caproni,*,1 Elaine Tritto,,1 Mario Cortese,* Alessandro Muzzi,* Flaviana Mosca,*
Elisabetta Monaci,* Barbara Baudner,* Anja Seubert,* and Ennio De Gregorio*
The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well
characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSK4)
adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in
mouse. Although all adjuvants boosted the Ab responses to TT, only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab
responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine, we investigated the gene signatures

Downloaded from http://www.jimmunol.org/ by guest on September 19, 2013

induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu
adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial
migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers
of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is
required for adjuvanticity to influenza vaccines, we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate
immunity pathways. By contrast, resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN
pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb, we confirmed that the adjuvanticity of MF59
and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent. The Journal of Immunology, 2012, 188: 30883098.

T
here are different types of influenza virus vaccines with
distinct immunogenicity profiles (13). Live-attenuated
virus vaccines induce a subclinical infection and there-
fore are very efficacious (4). Formalin-inactivated whole-virus
vaccines (WV) contain viral RNA, which activates IFN type I
responses that are required to boost the adaptive immune re-
sponses (5). Subunit virus vaccines contain hemagglutinin (HA)
and neuraminidase Ags, next to some residual structural proteins
(M1 and NP), but lack most of the viral RNA, except from some
residual RNA that may be associated with NP (6). These vaccines
are safer compared with WV, but they are also less immunogenic
and therefore require an adjuvant when administered to previously
unexposed or immunocompromised subjects. It has been proposed
that the best strategy to boost subunit flu vaccine formulation is to
restore IFN type I-associated activities that are lost during the
purification process. Accordingly, coadministration of type I rIFN
with a subunit influenza vaccine in mice was shown to increase
*Research Center, Novartis Vaccine and Diagnostics, 53100 Siena, Italy; and
protection against virus challenge (7). Alternatively, subunit flu
vaccine formulation immunogenicity could be restored by incor-
poration of IFN inducers such as the TLR9 agonist (5).
Clinical experience has shown that the oil-in-water emulsions are
extremely efficient in increasing the immunogenicity of flu subunit
vaccines. The adjuvant MF59 is an oil-in-water emulsion composed
of small droplets of squalene surrounded by a monolayer of nonionic
detergents (8, 9). Different aspects contribute to MF59 adjuvanticity.
Several studies have shown that MF59 can increase Ag uptake by
APCs (1012) besides inducing the activation of innate immunity
(13). In vitro studies have shown that MF59 induces the secretion of
chemokines in human macrophages, monocytes, and granulocytes
(14). We have previously demonstrated that i.m. injection of MF59
induces the local activation of innate immune reactions leading to
recruitment of blood cells in the muscle (13). Recently, it has been
shown that all the recruited cell types can take up both Ag and
adjuvant and that MF59 promotes the migration of Ag/adjuvant
double-positive cells to the draining lymph nodes (LNs) (15).
A MF59-adjuvanted influenza vaccine was developed initially
for seasonal vaccination in the elderly to enhance Ab titers and

Preclinical Safety Genomics, Novartis Institute of Biomedical Research, CH-4057
Basel, Switzerland
1
E.C. and E.T. contributed equally to this study.
Received for publication June 17, 2011. Accepted for publication January 13, 2012.
The microarray data presented in this article have been submitted to the ArrayExpress
database, European Molecular Biology Laboratory-European Bioinformatics (http://
www.ebi.ac.uk/), under accession number E-MTAB-942.
Address correspondence and reprint requests to Dr. Ennio De Gregorio, Novartis
Vaccine and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy. E-mail address:
ennio.de_gregorio@novartis.com
The online version of this article contains supplemental material.
Abbreviations used in this article: DC, dendritic cell; HA, hemagglutinin; HI, hem-
agglutination inhibition; KO, knockout; LN, lymph node; TT, tetanus toxoid; WV,
whole-virus vaccine.
Copyright 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
seroconversion and seroprotection rates (9). More recently it was
shown that also in young children MF59 enhances seroconver-
sion and cross-protection against flu strains mismatched to the
Ags of a seasonal subunit flu vaccine (16). MF59 and a second
oil-in-water emulsion, called AS03, have also been successfully
used for the development of prepandemic flu vaccines to protect
from a potential H5N1 pandemic influenza outbreak (17, 18). The
clinical data have shown that MF59 increases cross-reactive neu-
tralizing Ab titers and memory responses to H5 (17, 19). Recently,
MF59 and AS03 have also been used during the vaccination cam-
paign in the 20092010 H1N1 pandemic outbreak (20).
The ability of emulsions to increase the immunogenicity of sub-
unit flu vaccines seems to be quite unique. Clinical trials have shown
that the administration of MF59-adjuvanted H5N1 induces higher

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101764
The Journal of Immunology
hemagglutination inhibition (HI) and microneutralization titers to
flu compared with the administration of the same vaccine formulated
with alum (21). Furthermore, mouse studies have shown that MF59
enhances HI titers to seasonal flu Ags compared with alum and CpG
(22). However, the innate immune pathways required for the adju-
vant effect of oil-in-water emulsions to influenza subunit vaccines
and the exact role played by IFN type I are not well characterized.
In this work, we investigated the innate immune reactions that are
associated with adjuvanticity to influenza subunit vaccines in mouse
using a systems biology approach. First, we monitored the ability of
MF59, alum, and three TLR-dependent adjuvants, CpG, resiquimod,
and Pam3CSK4, to increase the immunogenicity of a commercial flu
seasonal subunit vaccine and to tetanus toxoid (TT). We found that
despite the fact that all adjuvants were able to increase the immu-
nogenicity of TT, only MF59 and to a less extent Pam3CSK4 were
able to adjuvant the flu vaccine. Then, we analyzed the innate
immune signatures induced by each adjuvant in vitro in splenocytes,
and in vivo at the injection site (muscle) and in the draining LNs
using DNA microarrays. Surprisingly, we found that the activation
of IFN type I pathway is not required for the adjuvanticity of MF59
and Pam3CSK4. In contrast, we found that despite a potent acti-
vation of IFN type I responses, resiquimod did not trigger any
adjuvant effect to the flu vaccine. Ultimately, we identified a gene
signature at the injection site that correlates with adjuvanticity to
influenza subunit vaccine.
Materials and Methods
Adjuvants
MF59 is a Novartis proprietary oil-in-water emulsion consisting of the 5% oil
squalene with the two surfactants, 0.5% Tween 80 and 0.5% Span-85, in
a sodium citrate buffer (10 mM). The alum used in this study is aluminum
hydroxide (Al-OH3). The CpG oligonucleotide sequence used was 59-TCC
ATG ACG TTC CTG ACG TT-39 with all phosphothioate backbones (CpG
1826). R848 (resiquimod) is an imidazoquinoline resuspended at 100 mM in
100% DMSO. Pam3CSK4 (Invivogen) is synthetic triacylated lipoprotein
resuspended at 1 mg/ml in MilliQ water.
Antigens
Trivalent flu Ags present in the 2007/2008 seasonal flu vaccine formulation
used in this study were egg-derived H1N1 (A/Brisbane/59/2007 H1N1-like
virus), H3N2 (A/Brisbane/10/2007 H3N2-like virus), and B (B/Florida/4/
2006-like virus). TT Ag was prepared and purified in house. All Ags were
diluted in PBS.
Mice and immunizations
Pathogen-free female BALB/c mice 68 wk of age were housed and treated
according to internal animal ethical committee and European guidelines.
In all experiments, mice were injected i.m. in both quadriceps muscles
with 50 ml dose per quadriceps with PBS, MF59 (1:1 dilution in PBS), 200
mg alum, 20 mg CpG (all in PBS), 1% DMSO in PBS, 25 mg R848, 10 mg
Pam3CSK4 (both in PBS, 1% DMSO), flu (a mix of H1N1, H3N2, and B
Ags; 0.1 mg each), and flu plus MF59. For adjuvanticity experiments, mice
were injected with the aforementioned adjuvants in presence or absence of
either 0.1 mg TT Ag or a mix of H1N1, H3N2, and B Ags (0.1 mg each).
Mice were immunized at days 0 and 21. Sera were collected 2 wk after
the first and the second immunization for Ab analysis. In Ab-blocking
experiments, mice were injected i.p. with 50 ml 20 mg antiIFN-ab R2
(Space Import-Export; bulk) or isotype control (goat IgG; R&D Systems)
1 d before and 7 d after the first immunization. Frequencies of influenza-
specific T cells in the spleens of four mice/group were measured 2 wk after
second immunization. T cell responses in LNs were measured on pools of
four mice/group 3 wk after the second immunization. For microarray and
confocal microscopy experiments, mice were sacrificed 6 h after treatment
and quadriceps muscles and inguinal LNs were collected. For flow cyto-
metric analysis of muscle cell composition or inguinal LN cell activation,
mice were sacrificed 24 h after treatment.
Ag-specific Ab measurement
Sera from mice injected with Ag with or without adjuvant twice were
collected 2 wk post-second immunization, and flu- or TT-specific Abs were
3089
measured by endpoint ELISA. MaxiSorp 96-well plates were coated with
2.5 mg/well flu Ag or 0.2 mg/well TT Ag in 100 ml/well PBS. Goat anti-
mouse IgG (Southern Biotechnology Associates), goat anti-mouse IgG1
(Southern Biotech), or goat anti-mouse IgG2a (Southern Biotechnology
Associates) Abs, all conjugated with alkaline phosphatase, were used at
1:2000 dilution. The reaction was developed by addition of 100 ml/well
p-nitrophenyl phosphate disodium salt substrate (Sigma-Aldrich), and ab-
sorbance at 405 nm was measured by Spectramax plate reader. ELISA
titers were expressed as the reciprocal dilution that gave an OD higher than
the average OD of the blanks plus 3 times the SD.
Measurement of flu HI inhibition titers
Sera were pretreated with DENKA receptor-destroying enzyme (Bio-
genetics) and then inactivated at 56C for 30 min. Two-fold serial
dilutions of 25 ml pretreated sera and positive control sera were treated
with 25 ml virus working dilution. Samples were incubated at room
temperature; 50 ml turkey RBC suspension was dispensed in each well;
and plates were again incubated at room temperature. The test endpoint
was determined by visual inspection for an agglutination reaction: a red
dot formation indicated a positive reaction (inhibition), whereas a diffuse
patch of cells a negative reaction (hemagglutination). The titer was de-
fined as the highest serum dilution at which hemagglutination was in-
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hibited, and the Ab concentration corresponds to the reciprocal value
of the titer.
In vitro splenocyte stimulation for microarray analysis
For microarray experiments on splenocytes, 106 cells/well were treated for
6 h with PBS, MF59 (at 1:300 dilution), 40 mg/ml alum, 1 mM CpG, 1%
DMSO, 5 mM R848, and 1 mM Pam3CSK4.
Muscle, LN, and splenocyte RNA extraction and purification
For muscle RNA extraction, whole muscles were homogenized in 7.5 ml
TRIzol (Invitrogen) with an Ultra-Turrax T25 (IKA), and total RNA was
extracted following the manufacturers protocol. One hundred micrograms
of RNA from each muscle was purified using the RNeasy RNA purification
columns (Qiagen) following the producers protocol. Residual DNA was
removed by an additional on-column DNase I digestion step using the
Qiagen RNase-free DNase set (Qiagen). RNA quality was assessed by
using the 2100 Bioanalyzer automated microfluidic system in combination
with the RNA 6000 Nano kit (Agilent) following the producers protocol.
For LN RNA extraction, the draining inguinal LNs from each mouse were
collected in liquid nitrogen and immediately homogenized on ice in lysis
buffer (Qiagen) using the Ultra Turrax T25 (IKA). The tissue lysates were
transferred in a QIAshredder column (Qiagen), and RNA was purified using
the RNeasy RNA purification columns (Qiagen) following the manufac-
turers protocol, as described before. RNA from splenocytes was extracted
using the Qiagen QIAshredder and RNeasy RNA purification kit (Qiagen)
following manufacturers protocol. For all microarray analyses shown in
this study, three independent experiments were conducted for each group.
RNA labeling, microarray hybridization, and data analysis
For each hybridization, 400 ng RNA was labeled using the QuickAmp
labeling kit (Agilent). Muscle or LNs RNA were labeled with cyanine-3 dye
for one-color microarray analysis. RNA from splenocytes treated with
adjuvants were labeled with Cy5 and cohybridized with cyanine-3labeled
RNA extracted from control buffer-treated splenocytes (reference) for two-
color microarray analysis. The efficiency of incorporation of the Cy3 or
Cy5 dyes was measured by Nanodrop analysis. A total of 1.65 mg labeled
cRNA was hybridized onto the Agilent 4 3 44k Whole Mouse Genome
Microarray chip, detecting .40,000 transcripts, for 17 h at 60C following
Agilent protocol. Images were acquired using the Agilent Scanner (Agi-
lent) at a resolution of 5 mm in extended dynamic range.
Raw images were preprocessed with the Agilents Feature Extraction 9.5
Image Analysis Software. Feature intensities and ratios were calculated,
and flags for bad quality were assigned to each spot. The BASE 1.2.17
database/analysis software was used for further data processing. Intensities
for each spot were normalized by the global median, and the local back-
ground was subtracted. The average ratio of the experimental replicates
was estimated, and aquantile normalization was applied. For multiple
hypothesis testing and type I error rate control, false discovery rate sta-
tistics based on the estimation of the q values for each t test were applied
(23). Significantly regulated probes were selected by using the following
cutoff: intensity ratio .4 (log2 ratio $|2|) and p value ,0.05 in at least one
treatment. Hierarchical clustering was performed with the TMEV 4.3.02
software on the log2 transformed dataset applying the Euclidean distance
matrix and the average linkage clustering method.
3090
Data deposition
The complete set of microarray data have been submitted to the ArrayExpress
database, European Molecular Biology Laboratory-European Bioinformat-
ics (ArrayExpress accession: E-MTAB-942, www.ebi.ac.uk/arrayexpress).
Correlation analysis between gene expression profiles in the muscle and IgG
titers was calculated as follows. First, the ratios of both gene expression in-
tensity levels and IgG titers in each animal induced by each adjuvant were
calculated against the corresponding nonadjuvanted formulations (MF59,
alum, and CpG against PBS, R848, and Pam3CSK4 against DMSO, respec-
tively). Then the ratio values were log2 rescaled, and averages and variances
were calculated. Finally, the linear correlation fit between gene expression and
IgG log2 ratios, weighted on both gene expression and IgG log2 ratio rescaled
variances, was evaluated. Supplemental Table II shows gene expression values
for the 1410 selected genes together with the correlation data between gene
expression profiles and influenza vaccine Ab titers for all the genes.
Confocal analysis of muscle cryosections
OCT-embedded muscle tissue was sectioned transversely at 5 mm, thaw
mounted onto Superfrost glass slides, and quickly air dried. Slides were
fixed in a 4% formaldehyde solution on ice and blocked in 3% BSA, 1%
saponin in PBS. Slides were then stained with goat anti-mouse utrophin
polyclonal IgG (Santa Cruz Biotechnology; 1:50 dilution) and rat anti-
mouse CD11b (AbD Serotec; 1:100 dilution), followed by a second-step
incubation with chicken anti-goat AlexaFluor 488 (Molecular Probes;
1:200 dilution) and goat anti-rat AlexaFluor 555 (Molecular Probes; 1:200
dilution) secondary Abs. Utrophin, a cytoskeletal protein playing a role in
anchoring the cytoskeleton to the plasma membrane and located in the
sarcolemma of muscle cells, was used to detect muscle fibers. CD11b/
Itgam was used as a marker for monocytes, macrophages, and granu-
locytes. Nuclei were detected using ToPro3 (Molecular Probes; 1:1000
dilution). Slides were mounted with ProLong gold antifade reagent (Invi-
trogen), coverslipped, and imaged on a LSM 710 microscope (Zeiss).
FACS analysis of LN single-cell suspensions
Inguinal LNs, excised from mice 24 h after adjuvant injection, were cut on ice;
resuspended in 0.5 ml RPMI 1640 medium (Life Technologies), 16 ml col-
lagenase D (Roche), and 25 ml DNase I (Roche); and incubated at 37C for
15 min under gentle agitation. The tissue was completely dissociated with
Gentlemax Dissociator (Miltenyi Biotec), followed by an additional 15-min
incubation at 37C. Cells were then resuspended in PBS, 5% FCS and
counted. A total of 1 3 106 cells/well was plated in duplicate, incubated for
20 min with 20% rabbit serum, and stained with two Ab combinations. For T,
B, and NK cells, a mix of antiDX-5 biotin (BD Pharmingen), anti-CD69
PerCP-Cy5.5 (eBioscience), anti-CD3 allophycocyanin-eFluor780 (eBio-
science), anti-CD19 A-700 (eBioscience), and Live/Dead Aqua (Invitrogen)
was used. For dendritic cells (DCs), anti-CD11c PerCP-Cy5.5, anti-CD86
allophycocyanin (Invitrogen), anti-MHCII A-700 (eBioscience), and Live/
Dead Aqua (Invitrogen) were used. AntiDX-5 biotin was detected with
a second-step incubation with streptavidin Pacific Blue (Invitrogen). Cells
were resuspended in FACS buffer before acquisition on a FACS LSRII flow
cytometer (BD Biosciences).
T cell cytokine response
To identify influenza-specific T cells, 1.5 3 106 cells/well were prepared
from spleens or pools of LNs excised from four mice/group, and stimu-
lated with 1 mg/ml anti-CD28 with a mix of H1N1, H3N2, and B Ags at
0.3 mg/ml total concentration. As a positive control, the same number of
cells was incubated with a mix of anti-CD3 and anti-CD28. As a negative
control, cells were incubated only with anti-CD28. After an overnight
stimulation, brefeldin A (2.5 mg/ml) was added for additional 4 h to inhibit
cytokine secretion. Cells were then stained with Live/Dead Aqua (Invi-
trogen), fixed, permeabilized with Cytofix/Cytoperm (BD Biosciences),
and then incubated with anti-CD16/CD32 Fc block (BD Biosciences).
Spleen-derived T cells were stained with the following mAbs: anti-CD3
PerCP-Cy5.5 (BD Pharmingen), antiCD4 V500 (BD Pharmingen), anti
IFN-g PE Green (BD Pharmingen), antiIL-2 allophycocyanin (BD
Pharmingen), anti-CD8 PE Texas Red (Invitrogen), antiIL-4 A488
(eBioscience), antiIL-13 A488 (eBioscience), antiTNF-a Alexa700 (BD
Pharmingen), antiIL-17 PE Cy7 (eBioscience), and antiCD44 V450 (BD
Pharmingen). LN-derived T cells were incubated with the following mAbs:
anti-CD3 PerCP-Cy5.5 (BD Pharmingen), anti-CD4 PE-A610 (Invi-
trogen), antiIFN-g PE (BD Pharmingen), antiTNF-a A700 (custom; BD
Pharmingen), antiIL-2 allophycocyanin (custom; BD Pharmingen), anti-
CD44 Horizon V450 (BD Pharmingen), and antiIL-4 A488 (BD Phar-
mingen). Cells were then acquired on LRSII (BD Biosciences) and ana-
lyzed using FlowJo software. Frequencies of influenza-specific T cells
INNATE IMMUNE SIGNATURES OF VACCINE ADJUVANTS
were calculated by subtracting the background measured in the corre-
sponding negative control for each cytokine.
Quantitative real-time PCR
The expression levels of five genes (Cxcl10, Il6, Ccl2, Tnfa, Mx1) induced
by injection of PBS, MF59, alum, and CpG (three mice for each group)
were measured by quantitative RT-PCR using the same RNA samples
previously used for microarray analysis. A total of 400 ng RNA from each
muscle was used for RT-PCR using qScript cDNA SuperMix kit (Quanta
Biosciences), according to the manufacturers protocol. Quantitative RT-
PCR was performed for each cDNA sample in duplicate using Perfecta
qPCR FastMix, UNG, Low Rox mix (Quanta Biosciences), and Applied
Biosystems TaqMan probes (Cxcl10, IL6, Ccl2, TNFa, Mx1) in Stratagene
MX3000P System. The thermal cycling program was as follows: an initial
10 min at 45C and 3 min at 95C, followed by 40 cycles of 15 s at 95C
and 1 min at 60C. The expression level of Gapdh was used as the internal
control to calculate the D cycle threshold.
Results
The efficacy of vaccine adjuvants is Ag dependent: MF59 and
Pam3CSK4 are the best adjuvants for a trivalent flu subunit
Downloaded from http://www.jimmunol.org/ by guest on September 19, 2013
vaccine
To assess how different classes of adjuvants enhanced Ab titers to
a coadministered Ag, BALB/c mice were immunized i.m. with TT or
with seasonal flu Ags (flu) either alone or in combination with the
following adjuvants: the oil-in-water emulsion MF59 and the mineral
salt alum (both TLR-independent adjuvants), the oligonucleotide
CpG, the small molecule resiquimod (R848), and the lipopeptide
Pam3CSK4 (agonists of TLR9, TLR7/8, and TLR2, respectively).
Two weeks post-second immunization, the Ab titers to both Ags and
the HI titers to flu were measured. PBS-injected mice were used as
control group for MF59, alum, and CpG, whereas 1% DMSO-treated
mice were the control group for R848 and Pam3CSK4. MF59 and, to
a lesser extent, Pam3CSK4 significantly enhanced total IgG titers to
H1N1, H3N2, and B Ags (Fig. 1A). The HI titers reflected what was
observed for total IgG titers: MF59 and Pam3CSK4 were the only
adjuvants that induced significantly enhanced HI titers to H3N2 (Fig.
1B). Isotype analysis of Ab titers showed that MF59 increased spe-
cific IgG1 and, to a lesser extent, IgG2a for all influenza Ags (Fig. 1C,
1D). Pam3CSK4 enhanced IgG2a and, to a lesser extent, IgG1 only in
response to H3N2. R848 and alum were not effective adjuvants for flu
Ags except for a modest effect of R848 on H3N2 IgG2a response.
CpG did not boost flu total Abs and HI titers, but induced a significant
increase of IgG2a titers (Fig. 1D). Consistently with the strong effect
of MF59 on IgG1 responses to influenza vaccine, we found that
MF59 was the only adjuvant that significantly increased Ag-specific
IL-4+/IL-13+ CD4 T cells in the spleen (Fig. 1E). To better evaluate
the quality of the CD4 response, we extended the analysis to other
cytokines. We found that the CD4 response to MF59-adjuvanted
vaccine was dominated by IL-4+/IL-13+ cells; however, we could
also detect high frequencies of IL-17+ and IL-2+ CD4 T cells and
CD4 T cells expressing multiple cytokines (Fig. 1F). We found that
CpG and Pam3CSK4 decreased the frequencies of Th2 (IL-4+) CD4
T cells in agreement with the known Th1 immunomodulatory effect
of TLR agonists (Fig. 1E). Alum and all the TLR agonists tested
failed to enhance influenza-specific CD4 responses (Fig. 1F). The
data produced in these experiments in animal model reflect clinical
data showing that MF59 is a very efficient adjuvant for subunit in-
fluenza vaccines, whereas alum is ineffective (21).
Differently to what was observed for flu, all adjuvants signifi-
cantly induced TT-specific total IgG titers (Fig. 2A). These data
show that all compounds tested in this study could act as adju-
vants, but their efficacy depends on the nature of the coadminis-
tered Ag. The analysis of Ab isotypes showed that MF59 and
Pam3CSK4 were the best inducers of IgG1 (Fig. 2B), whereas all
TLR agonists gave a superior IgG2a response (Fig. 2C).
The Journal of Immunology 3091

FIGURE 1. Humoral and cellular responses to influenza subunit vaccine formulated with different adjuvants. BALB/c mice (8 mice/group) were treated Downloaded from http://www.jimmunol.org/ by guest on September 19, 2013
with seasonal trivalent influenza Ags (flu) in combination with MF59, alum, CpG, Pam3CSK4, or R848, or in their respective control buffer (PBS or 1%
DMSO). Ab titers or HI titers were measured 2 wk post-second immunization. The data are representative of two independent experiments. (A) Total IgG
titers to flu Ags B Florida, H1N1 Brisbane, and H3N2 Brisbane. (B) HI titers to H3N2 Ag: the black circles represent values from single mice, and the red
bars represent the geometric mean titers over eight mice. (B) H1N1- and H3N2-specific IgG1 titers (C) and IgG2a titers (D) were measured 2 wk post-
second immunization. (E and F) Analysis of CD4 T cell responses to trivalent influenza Ags in combination with the various Ags in the spleen 3 wk post-
second immunization. (E) The graph shows the average frequencies of IL-4+/IL-13+ CD4 T cells from four mice/group as a percentage of total CD4 T cells.
(F) Frequencies of influenza-specific CD4 T cells expressing single (IFN-g, IL-4, IL-2, IL-17, or TNF-a) or different combinations of two (double) or three
(triple) cytokines. For Abs and T cell responses, unpaired two-tailed Student t test was performed comparing each group of mice treated with adjuvanted
vaccines with the corresponding nonadjuvanted control group. *p # 0.05.

MF59 is the strongest activator of cytokine genes in the muscle
and induces early recruitment of CD11b+ cells at the injection
site
To identify early innate immune gene signatures that correlate with
flu adjuvanticity, we analyzed the transcriptional profiles of mouse
splenocytes (in vitro), mouse muscles, and draining LNs (in vivo)
after treatment with the same adjuvants tested in the previous
experiments by whole genome microarray analysis. In a previous
kinetic study, we followed the transcription profile between 3 h and
4 d induced by several vaccine adjuvants after i.m. injection. We
found that most of the selected early innate immunity genes were
significantly regulated in the 6-h time point; therefore, we selected
the 6-h time point for all the microarray experiments performed in
this study (13). Significantly regulated genes were selected with an
3092
FIGURE 2. Ab responses to TT vaccine formulated with different ad-
juvant. BALB/c mice (8 mice/group) were treated with TT Ag in combi-
nation with MF59, alum, CpG, Pam3CSK4, or R848, or in their respective
control buffer (PBS or 1% DMSO). Total Ab titers (A), IgG1 (B), and
IgG2a (C) were measured 2 wk post-second immunization. Unpaired two-
tailed Student t test was performed comparing mice treated with adju-
vanted vaccines versus nonadjuvanted control group. *p # 0.05.
average log2 ratio $2 compared with control groups and a p value
#0.05 calculated on the three replicates per treatment group. Func-
tional analysis of the adjuvant-regulated genes showed that in vitro
in mouse splenocytes only the three TLR agonists were able to
activate transcription of cytokine-encoding genes (Fig. 3A, Sup-
plemental Fig. 1A). As expected, all TLR agonists upregulated
proinflammatory (Tnf, Il6) and Th1 cytokine (Il12 and Ifng) genes
(Supplemental Fig. 1A). On the contrary, the TLR-independent
adjuvants MF59 and alum did not modulate the expression of cy-
tokine genes or any other pathway (Supplemental Fig. 1A, data not
shown), suggesting that MF59 and alum poorly activate immune
cells in vitro. In contrast, in vivo, MF59 was the strongest modulator
of transcriptional events at the injection site, activating 36 of the 43
INNATE IMMUNE SIGNATURES OF VACCINE ADJUVANTS
cytokine-encoding genes modulated in the dataset. As previously
described, MF59 induced a large number of chemokine genes in-
volved in cell recruitment processes (13). Moreover, MF59 was the
strongest inducer of Il1-related genes (Il1a, Il1b, Il1rn, Il1f9) and
was the only adjuvant that upregulated Th2 cytokines (Il5 and Il4)
and Il9 (Supplemental Fig. 1A). Surprisingly, the TLR7 agonist
R848 activated only a very small number of cytokine genes: Cxcl9,
Cxcl10, Cxcl11, Cxcl13, and Tnfsf10/Trail, all of them known to
respond to IFN type I signaling (Supplemental Fig. 1A). However,
differently from the muscle, R848 was the strongest modulator of
gene expression in the draining LNs, modulating 34 of the 36 se-
lected cytokine genes (Fig. 3A). R848 cytokine response in LNs
was characterized by a strong activation of IFN type I and II genes
(Ifna2, Ifna4, Ifna12, Ifnb1, and Ifng) and Th1 cytokine genes (Il12b).
Pam3CSK4 induced the expression of 12 cytokine genes (mainly
chemokines and IL-1 family cytokines) in the LNs, whereas all other
adjuvants did not have any activity on cytokine expression or on any
other pathway examined.
An unbiased analysis of the gene expression dataset highlighted
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that at 6 h MF59 also induced in the muscle genes involved in
leukocyte transendothelial migration (KEGG mmu04670) and
cytokine binding (Fig. 3B, Supplemental Fig. 1B). CpG and
Pam3CSK4 also regulated some of these genes, like the adhesion
molecule genes Itgam, Icam1, and Vcam1. These data suggest that
MF59 could lead to stronger cell recruitment from the bloodstream
to the injection site compared with the other adjuvants tested in
these experiments. Among the genes encoding chemoattractants or
adhesion molecules, CD11b/Itgam (marker of myeloid lineage)
was selected as a biomarker to investigate local cell recruitment
events 6 h after i.m. adjuvant injection by confocal immunofluo-
rescence analysis of muscle cryosections (Fig. 2C). Muscle fibers
were visualized by using an Ab specific for utrophin, a ubiquitous
protein located at the neuromuscular synapse and myotendinous
junctions that plays a role in anchoring the cytoskeleton to the
plasma membrane in muscle cells. Nuclei were visualized by ToPro
3, a cyanine nucleic acid stain that allows DNA detection. At 6 h
MF59 and, to a lower level, Pam3CSK4 could draw afflux of
CD11b+ cells at the injection site. The muscle infiltrate was con-
firmed and further characterized by flow cytometry of muscle
single-cell suspensions at 24 h after injection. MF59 induced a
strong recruitment of neutrophils, eosinophils, and monocytes,
following treatment, whereas Pam3CSK4 induced mainly neutro-
phil influx (Fig. 3D). The observed recruitment of CD11b+ cells at
the injection site correlates very well with the adjuvanticity of the
different adjuvants to flu Ags (Fig. 1). Indeed, correlation analysis
between the gene expression profiles, induced by the different
adjuvants and the flu IgG Ab titers elicited in the adjuvanticity
experiments, highlighted selectin P (Selp), a cell adhesion molecule
that mediates the interaction of activated endothelial cells or pla-
telets with leukocytes by mediating rapid rolling of leukocytes
over vascular surfaces during the initial steps in inflammation, as
one of the few strongly positive correlating genes (Supplemental
Table I). Selp, one of the genes belonging to the leukocyte trans-
endothelial migration cluster, was strongly modulated by MF59
and Pam3CSK4 in the muscle (Fig. 3B), further supporting the
idea that enhanced cell recruitment at the injection might result in
improved adjuvanticity to subunit influenza vaccine.
To dissect the contribution of either adjuvant or flu Ags to
changes to the muscle or splenocyte transcriptomes, we evaluated
in a separate experiment the expression profiles of the three flu Ags
alone, flu Ags in combination with the adjuvant (MF59 + flu), and
adjuvant alone (MF59). We found that the flu Ags did not impact
the basal transcriptional program of splenocytes in vitro (data not
shown) or of the mouse muscle (Fig. 4). Moreover, the combi-
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FIGURE 3. MF59 is the strongest modulator of cytokine genes in the muscle and induces early recruitment of CD11b+ cells. (A) Number of significantly
regulated cytokine genes in splenocytes (in vitro), skeletal muscle, and inguinal LNs 6 h after treatment with the adjuvants tested in this study. (B) Ex-
pression profile of significantly expressed genes belonging to the leukocyte transendothelial migration pathway (KEGG mmu04670) in the muscle 6 h after
treatment with the various adjuvants. Some genes, like Icam1, appear more than once in the cluster because they are detected by multiple unrelated probes
in the Agilent 44k Whole-Mouse Genome Array. Genes were selected for fold change $|4| and p # 0.05 in at least one condition (see Materials and
Methods). The data are shown in log2 scale ranging from 23, green, downregulated to +3, red, upregulated. (C) Confocal analysis of muscle cryosections
taken 6 h after treatment showing utrophin in green, CD11b+ cells in purple, and a nuclei in blue. Scale bar, 40 mm. Results are representative of three
independent experiments. (D) FACS analysis of cell composition in the muscle 24 h after treatment with various adjuvants. Myeloid lineage cell (CD11b+)
neutrophils were defined as Ly6G+, monocytes as Ly6Chigh, eosinophils as Ly6Glow/side scatterhigh, macrophages as F4/80+, and DCs as CD11c+/MHC
class IIhigh. The events are shown as average counts over three mice with the relative SD.

nation of Ag with the adjuvant did not alter the transcriptional
profile of the adjuvant alone.
R848 strongly modulates IFN-related genes in the muscle and
draining LNs
Analysis of genes encoding for components of the IFN signaling
pathway or for IFN-responsive genes revealed that both R848
and CpG strongly induced these genes either in vitro in mouse
splenocytes or in vivo in muscle (Supplemental Fig. 2A), in agree-
ment with the available data on TLR7 and TLR9 signaling pathways
(24, 25). Interestingly, in the draining LNs, R848 was the only
regulator of IFN-related genes (61 of the 62 significantly modulated
genes in this cluster), whereas none of the other adjuvants tested
had an impact on transcription of these genes (Fig. 5A). In addition
to cytokine and IFN pathways, R848 was also a selective inducer of
cell activation marker genes such as CD69, CD86, and CD40 (Fig.
5B). These data together with the cytokine data shown in Fig. 2 and
Supplemental Fig. 1A suggest that R848 could induce a broad ac-
tivation of the immune cells in the LNs. CD69 and CD86 were used
as biomarkers in multicolor flow cytometric stainings of LN cells
24 h after i.m. injection of the different adjuvants in the absence of
coadministered Ag. In agreement with the microarray data, R848
was the only compound able to induce an increased expression of
CD69 on T, B, and NK cells (Fig. 5CE, respectively) and of CD86
in B cells (Fig. 5F). Moreover, R848 increased the number of DCs
(CD11c+/CD11b+) expressing CD86 and high levels of MHC class II
molecules (Fig. 5G). In summary, R848 was able to strongly induce
the IFN pathway and cytokine responses in vitro, to activate the IFN
pathway at the injection site, and to strongly activate IFN and cy-
tokine pathways in LNs, leading to T, NK, and B cell activation and
to DC recruitment. Interestingly, even if the broad immunostimu-
latory activity of R848 correlated with TT adjuvanticity, it was not
sufficient to promote flu adjuvanticity. By contrast, MF59, which is
the best adjuvant for a flu subunit vaccine among the adjuvants tested
in this work, was unable to activate either splenocytes or LNs in vivo,
whereas it worked efficiently locally at the injection site.
To validate the microarray data, we have analyzed the expression
profile of five genes (Cxcl10, Il6, Ccl2, Tnfa, and Mx1) in muscle
injected with PBS, MF59, alum, and CpG by quantitative RT-
PCR. The data obtained with microarray and RT-PCR using the
same RNA samples were very similar for all the genes tested
(Supplemental Fig. 2B).
MF59 and Pam3CSK4 adjuvanticity to flu and TT is IFN type I
independent
Type I IFN was shown to be a powerful adjuvant for a subunit flu
vaccine formulation (6). Similarly to MF59, rIFN type I coad-
ministered to a subunit flu vaccine increased HI titers and pro-
tection against intranasal influenza challenge in the mouse model.
The effect was shown to depend on IFN type I receptor (IFN-RI),
3094
FIGURE 4. Flu Ags do not alter the expression profile induced by MF59
in the muscle. Expression profiles of mouse muscle 6 h after treatment with
the three flu Ags in PBS (flu), flu Ags in combination with MF59 (MF59 +
flu), and adjuvant alone (MF59). (A) Overview of significantly regulated
genes; (B) subset of genes encoding for cytokines hierarchically clustered,
as described in Supplemental Fig. 1. The log2 scale ranges from 26, green,
downregulated genes to +6, red, upregulated genes.
as the adjuvant effect of IFN type I on influenza subunit vaccine was
abolished in IFN-RI knockout (KO) animals (7). Our microarray
analysis highlighted that the tested adjuvants induced differentially
the IFN-related genes. CpG and R848 were the most potent activators
of IFN-dependent genes, whereas very few IFN genes were activated
by MF59, alum, or Pam3CSK4 (Supplemental Fig. 2). These data
would suggest that the adjuvanticity of MF59, Pam3CSK4, and alum
is largely IFN independent, whereas the adjuvanticity of R848 and
CpG depends on IFN. To verify whether a functional type I IFN
pathway is required for the mechanism of action of these adjuvants,
mice treated with anti-IFN type I receptor Ab (anti-IFNAR2) and
then immunized with Ag plus adjuvant were compared with immu-
nized mice treated with the control isotype Ab. All the adjuvants
were tested in combination with TT (Fig. 6A), whereas only MF59
and Pam3CSK4, the two adjuvants able to enhance flu immunoge-
nicity, were tested in combination with flu Ags (Fig. 6BH). Two
weeks post-second immunization, sera were analyzed for Ag-specific
Ab titers. Mice treated with anti-IFNAR2, MF59, and Pam3CSK4
were still able to induce strong TT-specific total IgG Abs; alum and
CpG adjuvanticity was partially inhibited, whereas Ab titers dropped
considerably when R848 was used as adjuvant (Fig. 6A). Mice
treated with anti-IFNAR2 and immunized with trivalent flu vaccine
together with MF59 or Pam3CSK4 produced total IgG titers to the
three flu Ags similarly to isotype Ab-treated control mice after pri-
mary immunization (Fig. 6BD). Moreover, the response to MF59-
INNATE IMMUNE SIGNATURES OF VACCINE ADJUVANTS
adjuvanted vaccine was not affected by anti-IFN type I receptor Ab
treatment even after 2 wk from the second dose (Fig. 6EG). Similar
data were obtained for IgG1 and IgG2a isotypes (data not shown).
Surprisingly, the Ab response to H3N2 and B Ags adjuvanted with
Pam3CSK4 was enhanced by IFNAR2 Ab, suggesting that IFN type I
may have an inhibitory effect on the adjuvanticity of TLR2 agonists
after the booster dose. In parallel to the Ab response, we assessed the
Ag-specific T cell response in cells derived from the draining LNs by
intracellular cytokine staining 3 wk after the second immunization.
The presence of the anti-IFNAR2 Abs did not reduce the total
number of influenza-specific CD4 T cells induced by MF59 or
Pam3CSK4 (Fig. 6H). In the group treated with anti-IFNAR2 Ab
immunized with flu plus Pam3CSK4, we detected an increase of
IL-4+ CD4 T cells and a decrease of IFN-g+ CD4 T cells, suggesting
that IFN type I is involved in modulating Th1/Th2 balance in re-
sponse to TLR2 agonist.
Discussion
In the current work, we compared MF59 with alum and with the
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TLR-dependent adjuvants, CpG, R848, and Pam3CSK4 for their
ability to boost adaptive responses to flu subunit vaccine and to TT.
Although all the adjuvants were able to induce good Ab titers to TT,
only MF59 and, to a lower extent, Pam3CSK4 significantly in-
creased the humoral response to flu Ags. We cannot explain why HA
and TT have different requirements for adjuvanticity based on the
existing data. One possibility is that HA and TT interact differen-
tially with the various adjuvants used in this study. Indeed, it has been
shown that Ag-adjuvant codelivery can have a significant impact
on adaptive immune responses (26). In addition, the ability of HA to
bind sialic acid, a saccharide present in most human cell types, may
affect its biodistribution after i.m. injection and its ability to reach
the draining LNs. This may favor the adjuvants that exert a strong
local response, such as MF59 and Pam3CSK4, and not the adjuvants
that activate innate immunity directly in the LNs, such as R848.
The analysis of Ab subclasses in response to TT and influenza
showed that TLR agonists induced a Th1-biased response. MF59
induced a mixed response, even though the IgG1 to IgG2 ratio was
shifted toward IgG1, confirming that MF59 is an inducer of Th2-
biased responses in BALB/c (22, 27), a mouse strain genetically
skewed toward Th2 responses (28).
In agreement with Ab responses to influenza Ags, we found that
MF59 was a strong inducer of IL-4+/IL-13+ CD4 T cells known to
exert B helper function for production of IgG1 Abs. Despite the
ability of Pam3CSK4 to enhance the Ab response to influenza
vaccine, we could not measure a parallel increase of flu-specific
CD4 T cells. We suggest that Pam3CSK4 may have a modest
effect on CD4 T cells that we cannot measure with the sensitivity
of our current T cell assay.
Interestingly, the data obtained in our mouse model are con-
sistent with clinical data showing that MF59 can boost the re-
sponses to flu vaccines better than alum in humans (21). With the
exception of alum, we have investigated the effect of the various
adjuvants by simply mixing them with flu and TT. Therefore, we
cannot exclude that R848 and CpG would boost flu subunit vac-
cine responses if physically linked to the HA-Ag through direct
conjugation or through a delivery system. Indeed, several pub-
lications have shown that direct conjugation of TLR2, 7, and 9
agonists to the Ag increases their adjuvanticity (26, 29, 30).
In a first attempt to determine whether differential adaptive re-
sponses to flu correlated to distinct innate immune reactions in-
duced by the different adjuvants, we analyzed the transcriptional
profiles induced by each adjuvant in mouse splenocytes.
Consistently with previous observations, TLR-dependent adju-
vants induced strong transcriptional changes (31). Indeed, TLR
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FIGURE 5. R848 is the strongest modulator of IFN pathway in muscle and LNs and induces LN cell activation. (A) Number of significantly regulated
IFN-related genes in splenocytes, skeletal muscle, and inguinal LNs 6 h after treatment with various adjuvants. (B) Microarray expression of selected cell
surface-expressed genes in the draining LNs 6 h after treatment with the various adjuvants. (CE) Multicolor flow cytometric analysis of CD69 on T cells
(C), B cells (D), and NK cells (E) isolated from inguinal LNs 1 d after i.m. injection with the different adjuvants. (F) Multicolor flow cytometric analysis of
CD86 on B cells isolated from the draining LNs 1 d after i.m. injection with the different adjuvants. (CF) Data are shown as mean fluorescence intensity
(MFI) of either CD69 or CD86 on the relative subsets. (G) Multicolor flow cytometric analysis of MHC class II expression on DCs isolated from the
draining LNs 1 d after i.m. injection with the different adjuvants. The data represent the number of DCs expressing CD86 and high level of MHC class II
molecules after each treatment. SD bars were calculated over at least three independent experiments.

agonists rare known to directly activate splenocytes by binding to
TLRs. By contrast, MF59 and alum were not able to modulate blood
cell transcription in vitro, even though it has been shown that MF59
and alum induced secretion of chemokines in monocytes (14), and
that alum activated the NOD-like receptor protein 3 inflammasome
complex (32). We hypothesize that the activation of chemokine
secretion by blood cells in vitro by alum and MF59 is driven by post-
transcriptional events. More work needs to be done to investigate
whether alum and MF59 stimulate mRNA translation or secretion of
preformed chemokines. Next, we analyzed the transcriptional pro-
files induced by each vaccine adjuvant in the muscle and in draining
LNs. Even if MF59 did not activate innate immunity in vitro, it was
the strongest inducer of transcriptional changes at the injection site.
In particular, MF59 was the most potent inducer of genes coding for
chemokines, chemokine receptors, and transendothelial migration
factors. In agreement with microarray data, we detected a significant
infiltration of CD11b+ cells into the muscle 6 h after injection of
MF59. CD11b+ cell recruitment at 6 h was observed also after
Pam3CSK4 injection. Further characterization of muscle single-
cell suspensions by flow cytometry of the muscle 1 d after injec-
tion revealed that the cell populations recruited by MF59 and
Pam3CSK4 differed greatly. MF59 induced significant recruitment
of granulocytes, monocytes, and DCs, whereas Pam3CSK4 could
only induce neutrophil infiltration, which can explain some of the
differences in the quality of the immune responses elicited by these
two adjuvants. Overall, these results are in agreement with results
published by Mosca et al. (13) and by Calabro et al. (15) on the
effect of MF59 on muscle transcriptome and cell recruitment. The
striking correlation between the early cell recruitment events in the
muscle and the ability of the vaccine adjuvants to boost adaptive
response to HA suggests that leukocyte migration to the injection
site promotes adjuvanticity to flu subunit vaccines. Of course, these
3096 INNATE IMMUNE SIGNATURES OF VACCINE ADJUVANTS

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FIGURE 6. MF59 and Pam3CSK4 adjuvanticity to flu and TT is IFN independent. BALB/c mice (6 mice/group) were injected i.p. with antiIFN-ab
receptor 2 Ab (anti-IFNAR2) or control (iso), 1 d before and 7 d after the first immunization and immunized with TT or seasonal trivalent flu Ags either
alone or in combination with various adjuvants. (A) Two weeks post-second immunization, sera were analyzed for total IgG titers to TT. Two weeks post-
first immunization, sera were analyzed for total IgG titers for H1N1 (B), H3N2 (C), and B Florida (D). Two weeks post-second immunization, IgG titer sera
were analyzed for total IgG titers to H1N1 (E), H3N2 (F), and B Florida (G). The values are shown as mean titer over six mice. Unpaired two-tailed Student
t test was performed comparing mice treated with IFNAR2 versus mice treated with control Ab, *p # 0.05, **p # 0.01. (H) CD4+ T cell responses to
trivalent influenza Ags were measured 3 wk post-second immunization from a pool of four draining LN/group. The histograms show the percentage of
CD44+CD4 T cells positive for any cytokine. -, Unstimulated T cells; 3 mg, T cells stimulated with 0.3 mg/ml flu Ags. The percentage of CD4 T cells
positive for each cytokines following stimulation with flu Ags is shown in the graph below each group.

events are also linked to local reactogenicity of the vaccine. Inter- the muscle. Probably the blood cell types present in the muscle
estingly, R848 was able to strongly activate both IFN and cytokine respond differently to TLR7 activation compared with mouse blood
pathways in splenocytes, but induced only IFN-responsive genes in cells in vitro.
The Journal of Immunology
The substantial differences between splenocytes and muscle
transcriptional profiles in response to MF59, alum, and R848 show
that in vitro assays are not predictive of adjuvant activity in vivo.
In contrast to the injection site, MF59 had no impact on the
transcriptional activity of the draining LNs, whereas the TLR7
agonist R848 was the strongest modulator of LN transcription. Both
the cytokine/chemokine and the IFN-related gene clusters were
strongly induced by R848 in the draining LNs. Moreover, flow
cytometric analysis of LN single-cell suspensions showed that
R848 could activate T, B, and DCs. Because the adjuvants were
administered without Ags, the observed activation of both APCs
and adaptive immune cells in LNs by R848 is Ag independent,
and may not increase specific adaptive responses when R848 is
coadministered with HA. In addition, R848 may even trigger the
activation of autoreactive B and T cell clones. In a recently pub-
lished article, Koyama et al. (5) demonstrated that WV influenza
vaccines require TLR7-mediated IFN type I response for immu-
nogenicity. Moreover, rIFN-a, when administered at the time of
vaccination, was shown to act as a powerful adjuvant for a flu
subunit vaccine inducing protection against influenza virus chal-
lenge (7). Microarray and adjuvanticity data observed in our ex-
perimental settings would, however, suggest that IFN type I
activation can be dispensable in making a good adjuvant to flu
vaccine. Indeed, MF59 and Pam3CSK4 are not good inducers of
IFN-related genes, but are efficient adjuvants for the influenza
vaccine. In addition, despite the ability of CpG to induce a strong
IFN response in the muscle and of R848 to trigger the IFN
responses in muscle and LNs, none of these adjuvants were able to
boost adaptive responses to subunit flu vaccination. Analysis of
adaptive immune responses in mice in which the IFN type I sig-
naling was inhibited by treatment with an IFN type I receptor-
blocking Ab further confirmed this hypothesis. The adjuvanticity
of R848 to TT was almost completely abrogated in the absence of
IFN type I signaling. These data are consistent with a recent study
showing that IFN type I is required for R848 adjuvanticity to OVA
(33). A partial effect of anti-IFNAR2 Ab treatment on TT adju-
vanticity was also found for alum and CpG, whereas Pam3CSK4
and MF59 were not significantly affected. Moreover, in the case of
flu Ags, the MF59- or Pam3CSK4-dependent increase in Ab titers
after first immunization was not affected by anti-IFNAR2, further
demonstrating that adjuvanticity of MF59 and Pam3CSK4 is IFN
type I independent. Our data on Ab titers and cellular responses
after the influenza vaccine boost confirmed that MF59 is inde-
pendent of IFN type I, and they suggest that IFN type I may even
play a regulatory role in response to Pam3CSK4 by modulating
the Th1/Th2 balance.
By contrast to our data, a previous study has shown that Ab
responses to MF59-adjuvanted flu are partially affected in an IFN-
RI KO background (7). These differences may be explained by the
fact that immune cells that develop in the absence of IFN-RI may
have an intrinsic defect to respond to vaccination that is not de-
pendent on the adjuvant used. Indeed, in the previous study by
Proietti et al. (7), the IFN type I KO mice had reduced Ab
responses also to a nonadjuvanted flu vaccine.
In summary, in this work we show that direct, Ag-independent
activation of innate immunity in the draining LN by vaccine
adjuvants is not required to boost adaptive responses to a flu subunit
vaccine. By contrast, an early, local recruitment of CD11b+ blood
cells at the injection site by MF59 and by the TLR2 vaccine ad-
juvant Pam3CSK4 strongly correlates with the ability of these
adjuvants to enhance Ab responses to flu Ags. Within this CD11b+
population, a higher frequency of professional APCs recruited by
MF59 compared with Pam3CSK4 correlated to an even more en-
hanced immune response to flu vaccine.
3097
Acknowledgments
We thank M. Tortoli and the Animal Care Facility at Novartis Vaccines
Siena for all mice experiments; C. Malzone for help on the analysis of
IgG titers; D. Casini for support with the ELISA experiments; and S. Nuti,
S. Tavarini, and C. Sammicheli for help on the FACS experiments.
Disclosures
All authors, with the exception of E.T., are employees of Novartis Vaccines
and Diagnostics. E.T. is employed by Novartis Institute of Biomedical Re-
search, in the same Novartis group.
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