AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity.
ABSTRACT Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-kappaB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4(+) T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. LPS elicits strong immunopathological responses during bacterial infection, and the lipid A moiety of LPS is responsible for this immunostimulatory activity. Lipid A exerts its biological activity by sending signals via TLR4 present on immune cells, and TLR4 agonists have been a target for vaccine adjuvant. Previously, we demonstrated an adjuvant activity of deacylated lipooligosaccharide (dLOS) to viral and bacterial antigens. In this study, we characterized the chemical structure of dLOS and evaluated its immunostimulatory activity on mouse and human immune cells in comparison with monophosphoryl lipid A (MPL). dLOS consists of a core oligosaccharide lacking the terminal glucose residue, a glucosamine disaccharide with two phosphate groups, and two N-linked acyl groups. dLOS was similar to MPL in induction of cytokine production in mouse peritoneal macrophages, but was a more potent activator in human monocytes and dendritic cells (DCs). Results of an analysis of allogeneic T cell responses revealed that dLOS induces Th1, Th2, and Th17-type immune responses in a dose-dependent manner. The immunostimulatory activities of dLOS were completely abrogated in TLR4(-/-) mice, which confirms its TLR4-dependency. These results suggest that in the presence of the core oligosaccharide, O-linked acyl groups of LPS are dispensable for activating the TLR4 signaling pathway. dLOS did not cause any pathological effects or death at 0.25, 0.5, or 1 mg per kg body weight in mice in the acute toxicity tests. This result suggests that dLOS has a low toxicity. dLOS should be considered for further development as a safe and effective adjuvant for human vaccines.PLoS ONE 01/2014; 9(1):e85838. · 3.53 Impact Factor
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ABSTRACT: Aluminum-adjuvanted vaccines are administered through an intramuscular injection (IM) in the US and EU, however, a subcutaneous injection (SC) has been recommended in Japan because of serious muscle contracture previously reported following multiple IMs of antibiotics. Newly introduced adjuvanted vaccines, such as the human papillomavirus (HPV) vaccines, have been recommended through IM. In the present study, currently available vaccines were evaluated through IM in mice. Aluminum-adjuvanted vaccines induced inflammatory nodules at the injection site, which expanded into the intra-muscular space without any muscle degeneration or necrosis, whereas non-adjuvanted vaccines did not. These nodules consisted of polymorph nuclear neutrophils with some eosinophils within the initial 48h, then monocytes/macrophages 1 month later. Inflammatory nodules were observed 6 months after IM, had decreased in size, and were absorbed 12 months after IM, which was earlier than that after SC. Cytokine production was examined in the injected muscular tissues and AS04 adjuvanted HPV induced higher IL-1β, IL-6, KC, MIP-1, and G-CSF levels in muscle tissues than any other vaccine, but similar serum cytokine profiles were observed to those induced by the other vaccines. Currently available vaccines did not induce muscular degeneration or fibrotic scar as observed with muscle contracture caused by multiple IMs of antibiotics in the past.Vaccine 04/2014; · 3.77 Impact Factor
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ABSTRACT: Recent reports that TLR4 and TLR7 ligands can synergistically trigger Th1 biased immune responses suggest that an adjuvant that contains both ligands would be an excellent candidate for co-administration with vaccine antigens for which heavily Th1 biased responses are desired. Ligands of each of these TLRs generally have disparate biochemical properties, however, and straightforward co-formulation may represent an obstacle. We show here that the TLR7 ligand, imiquimod, and the TLR4 ligand, GLA, synergistically trigger responses in human whole blood. We combined these ligands in an anionic liposomal formulation where the TLR7 ligand is in the interior of the liposome and the TLR4 ligand intercalates into the lipid bilayer. The new liposomal formulations are stable for at least a year and have an attractive average particle size of around 140 nm allowing sterile filtration. The synergistic adjuvant biases away from Th2 responses, as seen by significantly reduced IL-5 and enhanced interferon gamma production upon antigen-specific stimulation of cells from immunized mice, than any of the liposomal formulations with only one TLR agonist. Qualitative alterations in antibody responses in mice demonstrate that the adjuvant enhances Th1 adaptive immune responses above any adjuvant containing only a single TLR ligand as well. We now have a manufacturable, synergistic TLR4/TLR7 adjuvant that is made with excipients and agonists that are pharmaceutically acceptable and will have a straightforward path into human clinical trials.Journal of Nanobiotechnology 04/2014; 12(1):17. · 5.09 Impact Factor
of June 13, 2013.
This information is current as
Response Leading to Enhanced Adaptive
Transient Localized Innate Immune
Agonist-Based Adjuvant System, Induces a
AS04, an Aluminum Salt- and TLR4
Schiavetti, Daniel Larocque, Marcelle Van Mechelen and
Kielland, Olivier Vosters, Nathalie Vanderheyde, Francesca
Sandra L. Giannini, Michel Bisteau, Harald Carlsen, Anders
Arnaud M. Didierlaurent, Sandra Morel, Laurence Lockman,
2009; 183:6186-6197; Prepublished online 28
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Copyright © 2009 by The American Association of
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The Journal of Immunology
by guest on June 13, 2013
AS04, an Aluminum Salt- and TLR4 Agonist-Based Adjuvant
System, Induces a Transient Localized Innate Immune
Response Leading to Enhanced Adaptive Immunity1
Arnaud M. Didierlaurent,2,3* Sandra Morel,2* Laurence Lockman,* Sandra L. Giannini,*
Michel Bisteau,* Harald Carlsen,†Anders Kielland,†Olivier Vosters,4‡Nathalie Vanderheyde,*
Francesca Schiavetti,5* Daniel Larocque,§Marcelle Van Mechelen,* and Nathalie Garc ¸on*
Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4?-monophosphoryl lipid A) and aluminum salt. It is a new
generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine
Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04
was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this
period, AS04 transiently induced local NF-?B activity and cytokine production. This led to an increased number of activated Ag-loaded
dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells.
AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4?T or B lymphocytes. These AS04-induced
innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged
the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum
and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted
vaccines. The Journal of Immunology, 2009, 183: 6186–6197.
cines based on soluble recombinant Ags typically require adjuvants to
enhance an Ag-specific adaptive immune response, i.e., a T cell and
Ab response (1). The induction of an optimal innate immune response
is also associated with an enhanced adaptive immune response. In
light of the recent advances in understanding the receptors and asso-
ciated agonists responsible for the induction of the innate immune
response (e.g., TLRs), a new generation of adjuvants incorporating
these agonists has been engineered for new prophylactic and thera-
peutic vaccines (2). Understanding how adjuvants work at the mo-
lecular level and more importantly in vivo is critical for the develop-
ment of safe and more efficient vaccines.
Adjuvant System 04 (AS04)6consisting of MPL (3-O-desacyl-
4?-monophosphoryl lipid A) adsorbed onto a particulate form of
he development of new vaccines has highlighted the need
for effective and long-lasting protection. In particular, vac-
aluminum salt is one of these new generation adjuvants now li-
censed for use in humans (1). AS04 is currently a component in
two licensed vaccines, one against the cervical precancerous le-
sions and cancer containing virus-like particles (VLPs) of the L1
protein from human papillomavirus (HPV)-16 and HPV-18 onco-
genic strains of HPV (Cervarix) (3–5) and the second against hep-
atitis B virus (Fendrix) (6). A third vaccine against herpes simplex
2 virus is in phase III clinical trials.
MPL is a detoxified derivative of the LPS isolated from the
Gram-negative bacterium Salmonella minnesota R595 strain (7–
9). LPS has been found to function as a specific agonist of TLR4
(10, 11). MPL signals via TLR4 (12–14), although one report de-
scribes signaling via TLR2 (13). TLR4 stimulation can contribute
to the activation of the innate immune response, by activating
NF-?B transcriptional activity and the subsequent expression of
proinflammatory cytokines, such as TNF-? and IL-6 (15). These
cytokines can in turn enhance the adaptive immune response by
stimulating the maturation of APCs while repressing the tolerance
response through the inhibition of regulatory T cell activity (16).
MPL is generally reported to promote IFN-? production by Ag-
specific CD4?T cells, therefore skewing the immune response
toward a Th1 profile (9). A Th1 immune response is required for
effective protection against intracellular pathogens.
Aluminum salt has a long history of being used as an adjuvant
(17) and has been shown as having a bias toward promoting Abs
and a Th2 response. This type of immune response is effective
against extracellular pathogens, but not against intracellular patho-
gens. Due to its particulate nature, aluminum salt is considered to
act as a depot for vaccine Ag components, which enhances Ag
*GlaxoSmithKline Biologicals, Rixensart, Belgium;†Cgene, Oslo, Norway;‡Institute
for Medical Immunology, Universite ´ Libre de Bruxelles, Charleroi, Belgium; and
§GlaxoSmithKline Biologicals, Laval, Canada
Received for publication May 11, 2009. Accepted for publication September
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by GlaxoSmithKline Biologicals (to H.C. and A.K.).
2A.D. and S.M. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Arnaud Didierlaurent, Glaxo-
SmithKline Biologicals, Rue de l’Institut 98, 1330 Rixensart, Belgium. E-mail
4Current address: Institut de Recherche Interdisciplinaire en Biologie Humaine et
Mole ´culaire, Universite ´ Libre de Bruxelles, Brussels, Belgium.
5Current address: Novartis Vaccines, Sienna, Italy.
6Abbreviations used in this paper: AS04, Adjuvant System 04; VLP, virus-like par-
ticle; HPV, human papillomavirus; DC, dendritic cell; BMDC, bone marrow DC;
Fluo-OVA, Alexa Fluor 647-labeled OVA; HEK, human embryonic kidney; CBA,
cytokine bead array.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
by guest on June 13, 2013
uptake by APCs. At a molecular level, aluminum salt has been
found to stimulate Nlrp3, a component of the inflammasome (18–
20). The inflammasome functions as an intracellular multiprotein
platform for the recruitment and activation of caspase-1 and the
subsequent processing of proform of cytokines such as IL-1? or
IL-18. In vitro, this requires the pretreatment of APCs with TLR
ligands, such as MPL, as aluminum salt alone is reported not to
be able to induce the transcription of IL-1? or IL-18 genes.
There is therefore a theoretical basis to suggest that MPL and
aluminum salt synergize in AS04 to produce elevated levels of
IL-1? or IL-18. However, whether Nlrp3 and IL-1?/IL-18 play
a role in the adjuvant activity of aluminum salt alone is a matter
of debate (21).
In the Cervarix vaccine, AS04 is formulated with the hydroxide
salt of aluminum. The Ag component of the vaccine is made up of
VLP incorporating the major coat protein L1 from HPV-16 and
HPV-18 strains (1). Recent work has shown that AS04, compared
with an adjuvant containing only aluminum salt, induced a higher
and long-lasting immune response to identical VLP Ag compo-
nents of the respective HPV vaccines (22). Both AS04 and MPL,
but not aluminum salt alone, were found to induce TNF-? secre-
tion in monocytes suggesting that these in vivo differences in im-
munogenicity arose from the capacity of MPL to stimulate TLR4
signaling. Therefore the objective of this study was to assess the
contribution of MPL in AS04 response and whether the MPL-
specific response was modulated by its adsorption on aluminum
salt in the context of the Cervarix vaccine. To achieve this, the
response induced by the vaccine and its separate components were
investigated in vitro on human cells and in vivo in mice following
i.m. injection. In these experiments, the spatial and temporal pa-
rameters for AS04 to function as an efficient adjuvant were defined
and related to how the vaccine and its components stimulated
TLR4 signaling, cytokine expression, and APC activation.
Materials and Methods
A 50-?l dose of vaccine contained 5 ?g of clinical grade MPL (purified
from S. minnesota), 2 ?g each of HPV-16 and HPV-18 L1 VLPs Ags (for
preparation of the Ags, see Ref. 22) and 50 ?g of aluminum hydroxide
(Brenntag). The 50-?l vaccine dose used in this study represents one-tenth
of the routine Cervarix vaccination dose in human subjects. In some ex-
periments, 5 ?g of OVA (Calbiochem) or Alexa Fluor 647-labeled OVA
(Fluo-OVA; Invitrogen) was used instead of HPV L1 VLPs.
Stimulation of TLR-transfected human embryonic kidney (HEK)
Transfected HEK 293 cells (InvivoGen) with the expression vectors en-
coding 1) TLR4, MD-2, and CD14, 2) TLR1 and TLR2, or 3) TLR2 and
TLR6 were further stably transfected with the NF-?B reporter vector
pNifty-2 secreted alkaline phosphatase (Invivogen). Cells were cloned by
zeomycin (50 ?g/ml) with further selection on the basis of appropriate
TLR-stimulated expression of secreted alkaline phosphatase. Transfected
cell lines were plated at 2 ? 105cells per well in a 24-well plate cultured
in medium containing DMEM, 4.5 g/L glucose and L-glutamine, 10% FCS,
and 0.5% penicillin/streptomycin. Cells were stimulated 24 h later for 5 h
in FCS-free medium containing MPL and aluminum hydroxide as indicated
or with LPS (0.1 ?g/ml, from S. minnesota; InvivoGen), PAM2CSK4
(S)-lysyl-(S)-lysyl-(S)-lysine ? 3 CF3COOH; 5 ?g/ml; EMC Microcol-
lections), PAM3CSK4 (5 ?g/ml; EMC Microcollections), or PBS. Se-
creted alkaline phosphatase activity was measured using QUANTI Blue
Preparation and in vitro stimulation of human cells
PBMC were isolated from the buffy coat fraction obtained from routine
blood donor donations. Mononuclear cells were obtained by centrifugation
over Ficoll-Hypaque gradients. PBMCs were cultured in complete medium
containing RPMI 1640 supplemented with 50 ?M 2-ME, 1 mM sodium
pyruvate, 100 U/ml penicillin, 100 ?g/ml streptomycin, 2 mM L-glutamine,
1% nonessential amino acids (Life Technologies), and 5% FCS (PAA
Laboratories). To measure intracellular cytokine production in monocytes,
PBMCs at indicated concentrations were stimulated in triplicate in 96-well
plates at 37°C for 30 min before adding brefeldin A for an additional 5.5 h.
Cells were then washed and detached using 2 mM EDTA in PBS. After
surface staining with CD14 Abs, cells were labeled intracellularly for IL-6
and TNF-? and analyzed by flow cytometry. In some experiments, PBMCs
were preincubated for 1 h at 37°C with 2 ?g/ml blocking anti-TLR4 Ab
(clone HTA125; eBioscience), an anti-TLR2 Ab (clone TL2.1; eBioscience),
or both. Peptidoglycan from Staphylococcus aureus (FLUKA; Sigma-
Aldrich) was used as TLR2 agonist. For the evaluation of IL-1? pro-
duction, 1 ? 106PBMCs were stimulated in 96-well plates for 18 h and
supernatants were collected in triplicates. The caspase-1 inhibitor Y-
VAD (Calbochem) was used at 10 ?g/ml. Human IL-1? was measured by
cytokine bead array (CBA; BD Biosciences). Human DCs were prepared
from PBMC-derived monocytes treated with GM-CSF (100 ng/ml; R&D
Systems) and IL-4 (100 U/ml; PeproTech) for 6 days. Cells were resus-
pended in complete medium at 1 ? 106cells/ml and stimulated in 96-well
plates in triplicate for 18 h. IL-6 and TNF-? were measured in the super-
natant by CBA. The cells were further analyzed for CD83 and CD86 by
flow cytometry. CD4?T cells (from six donors) were purified from fresh
PBMC using a MACS column and a negative selection CD4?T cell iso-
lation kit (Miltenyi Biotec). The whole isolation procedure was conducted
at 4°C and purity of ?95% was confirmed by flow cytometry. Purified
CD4?T cells were cultured at 2 ? 105cells/200 ?l of RPMI 1640 with
10% FCS. Cells were stimulated for 18 h with 1 ?g/ml coated anti-CD3
(OKT3; Sigma-Aldrich) in the presence or absence of MPL (10 ?g/ml).
Brefeldin A (1 ?g/ml) was added for the last 16 h and the cells were
analyzed by flow cytometry after intracellular staining.
All experiments and assays were performed in accordance with the local
national Ethical Principles and Guidelines for Animal Experimentation.
Female C57BL/6, BALB/c mice were obtained from Harlan Horst. NF-
?B-luciferase transgenic reporter mice were bred in the Cgene facilities
(23). Female OVA-TCR transgenic mice DO11.10 strain (BALB/c back-
ground) and OT-II strain (C57BL/6 background) were provided by V.
Flammand (Institute for Medical Immunology, Charleroi, Belgium) and F.
Andris (Universite ´ Libre de Bruxelles, Charleroi, Belgium), respectively.
The i.m. injections were performed on either the gastrocnemius or tibialis
anterior in 50, 25, or 10 ?l depending on the experiment. The i.v. injections
(100 ?l) were performed in the tail vein.
Anti-HPV-16 L1 VLP and anti-HPV-18 L1 VLP ELISA
Anti-HPV-16 L1 VLP and anti-HPV-18 L1 VLP Ab titers were measured
by ELISA, following the protocol previously described (22), in serum sam-
ples taken from mice 14 days after first injection.
Measurement of luciferase activity in the NF-?B luciferase
NF-?B activation was monitored in transgenic NF-?B-luciferase mice in
which expression of luciferase is regulated by three NF-?B response ele-
ments. In vivo imaging of NF-?B activity was performed noninvasively or
in mice with surgically exposed lymph nodes following i.p. injection of
D-luciferin (120 mg/kg; Biosynth). Time from D-luciferin injection to im-
age acquisition was 10 and 25 min, respectively. Acquisition time was
typically 1–2 min using the IVIS100 Imaging System (Xenogen), com-
posed of a light-sealed imaging chamber fitted with a heating platform
(37°C) and cooled CCD camera (?105°C). Image acquisition and analysis
were done with the accompanying IVIS100 software. During the imaging
period, mice were anesthetized with 2.5% isoflurane. Immediately after in
vivo imaging, mice were killed by cervical dislocation and the dissected
spleen specimens were snap-frozen in liquid nitrogen. Individual frozen
organs were homogenized in reporter lysis buffer (Promega) and cleared by
centrifugation. Luciferase activity was quantified (conventional luminom-
eter; Turner Instruments) in the supernatant by adding luciferin, ATP, and
magnesium, following the manufacturer’s instructions (Promega). Lucif-
erase activity was normalized to protein content (Bio-Rad).
Measurement of cytokines in mice
Cytokines were measured in murine muscle and serum samples. For each
pooled muscle sample, injected gastrocnemius muscles were pooled and
homogenized using an Ultraturax (VWR) in 3 ml of PBS plus anti-protease
inhibitor mixture (Sigma-Aldrich). The homogenates were then cleared by
centrifugation at 14,000 rpm for 10 min, and supernatants were stored at
?70°C before analysis. Serum samples were measured from individual
6187The Journal of Immunology
by guest on June 13, 2013
mice. Cytokine levels were measured according to the manufacturer’s rec-
ommendations by flow cytometry using CBA for IL-6, IL-12, TNF-?, IL-
1?, CCL2, and CCL3 or by Meso Scale Discovery kit for CXCL1, IL-10,
IL-12, or IFN-?. IFN-? was measured by ELISA (PBL Biomedicals).
Preparation of mouse cells
Cell suspensions from the spleen and from the left and right axillary non-
draining lymph nodes from each individual mouse and from pooled iliac
lymph nodes were prepared using a Potter Homogenizer in medium (RPMI
1640, 10% FCS, 100 U/ml of penicillin, 100 ?g/ml streptomycin 2 mM
L-glutamine, 50 ?M 2-ME, 1 mM sodium pyruvate) and passed through a
100-?m filter. Cells were then washed and counted using a Multisizer
Measurement of OVA uptake
Bone marrow DCs (BMDCs) were prepared from bone marrow cells
treated with mouse GM-CSF (10 ng/ml; R&D Systems) for 6 days.
BMDCs were resuspended in complete medium at 1 ? 106cells/ml and
incubated in 96-well plates in triplicate with Fluo-OVA-containing formu-
lations at the indicated concentrations. Five hours later, the cells were
washed extensively and the level of Fluo-OVA uptake was analyzed by
flow cytometry. In vivo, mice (n ? 4; 4 pools of 6 mice/group) were
immunized i.m. with Fluo-OVA-containing formulations as indicated. Af-
ter 24 h, cells were prepared from pooled iliac lymph nodes and analyzed
by flow cytometry.
Measurement of Ag presentation to OVA-specific T cells
OVA-specific CD4?T cells were purified from pooled spleen and lymph
nodes of OVA-TCR transgenic DO11.10 and OT-II mice using a CD4 T
cell negative selection kit (Dynal Biotech) according to the manufacturer’s
recommendations. Purified CD4?T cells were then stained with CFSE.
Briefly, 1 ? 107T cells/ml were incubated with 5 ?M CFSE in RPMI 1640
for 10 min at 37°C. Cold RPMI 1640 plus 5% FCS was added to the cells
to stop CFSE labeling and the cells were washed extensively.
BMDCs from BALB/c mice (for DO11.10 T cells) and C57BL/6 mice
(for OT-II T cells) at 1 ? 106cells/ml were incubated in complete medium
with OVA-containing formulations at the indicated concentrations for 6 h
in 24-well plates and washed extensively. Lymph node CD11c?DCs were
purified using CD11c?selection kit (Miltenyi Biotec) from pooled iliac
lymph nodes from mice that were immunized 1 day before with OVA-
containing formulations (n ? 24 mice per vaccination). Treated BMDCs or
purified lymph node DCs were incubated with 1 ? 105CFSE-labeled
OVA-specific T cells in 96-well plates at the indicated DC to T cell ratio.
After 3 days, the cells were washed and the proliferation of the T cells was
assessed by flow cytometry.
Cells were resuspended in PBS, 1% FCS, and 1 mM EDTA and stained
with the Abs described. To measure activation of human DCs, cells
were stained using anti-CD86-FITC, anti-CD1a-PE, anti-CD83-allo-
phycocyanin. To evaluate intracellular cytokine production in human
monocytes, stimulated cells were surface stained for anti-CD14-FITC
and then permeabilized using the Cytofix/Cytoperm kit (BD Bio-
sciences). Intracellular cytokines were revealed by staining the cells
with anti-IL-6-PE and anti-TNF-?-allophycocyanin Abs. A similar pro-
tocol was used to measure T cell activation using anti-CD4-PE, anti-
CD69-allophycocyanin Cy7, and anti-CD40L-PE as surface markers and in-
tracellular anti-IFN-? FITC. To measure activation of murine immune cells,
cells were first treated with 2.4G2 Ab for 5 min to block the Fc receptor and
stained with the following Abs: anti-CD11b-Pe Cy7, anti-CD11c allophyco-
cyanin, anti-MHC class II biotin, CD40 PE and anti-CD86 FITC, and anti-
F4/80 biotin. To analyze Fluo-OVA uptake in iliac lymph node, the cells were
stained with anti-Ly6C FITC, anti-MHC class II biotin, anti-CD11b Pacific
blue, and anti-CD11c-Pe Cy7. To analyze T cell proliferation by CFSE dilu-
tion, the cells were stained with anti-CD4 allophycocyanin and the clonotypic
anti-OVA TCR Ab KJ1.26 PE (DO11.10) or the combination of anti-TCR
labeled streptavidin. All Abs were obtained from BD Biosciences. Fluorescent
events were acquired using an LSR2 and analyzed using FACSDiva software
Splenocyte stimulation by HPV-16 or HPV-18 L1 VLP Ags
Three groups of BALB/c mice (n ? 12) were immunized i.m. (days 0 and
21) with 2 ?g of HPV 16/18 L1 VLPs alone, or formulated with aluminum
hydroxide (50 ?g) or with AS04 (50 ?g of aluminum hydroxide plus 5 ?g
agonist. A, HEK cells transfected with
tlr4, md2, and cd14; with tlr1 and tlr2;
or with tlr2 and tlr6 were stimulated
(Alum), or both at indicated concen-
trations (?g/ml), PBS or positive con-
trols (? controls) in LPS (0.1 ?g/ml)
(top), PAM3CSK4 (5 ?g/ml) (mid-
dle), and PAM2CSK4 (5 ?g/ml) (bot-
tom). Cells were plated at 2 ? 105
cells/well in 24-well plates 24 h before
stimulation. Data represent the mean
relative levels of secreted alkaline
phosphatase activity as a measure of
NF-?B activity (in triplicate cultures).
Error bars describe SDs. B, The pro-
portion of TNF-?- and IL-6-positive
human CD14?monocytes in PBMC
cultures (5 ? 105cells/well; 96-well
plate) stimulated for 6 h with MPL
(0.1 ?g/ml), or peptidoglycan (PGN,
0.1 ?g/ml), in presence of medium
(control), an anti-TLR2- or an anti-
TLR4-blocking Abs. Data represent
geometric mean and the symbols rep-
resent the data points from each of the
donor samples tested (n ? 5 donors).
MPL acts as a TLR4
6188AS04 INDUCES A TRANSIENT LOCALIZED INNATE IMMUNE RESPONSE
by guest on June 13, 2013
MPL) in 50 ?l. Two weeks after the second immunization, spleen cell
suspensions were prepared from pooled mice (n ? 4, 4 pools of 3 spleen
samples/group). Cells were cultured at a final concentration of 5 ? 106
cells/ml in 1 ml per flat-bottom 24-well plates with 1 ?g/ml L1 HPV-16 or
HPV-18 Ag. Supernatants were harvested at 48 h later and tested for the
presence of IFN-?, IL-2, and IL-5 using a mouse CBA kit.
Statistical analyses were performed on logarithmic transformed data. The
Shapiro-Wilk test was used to confirm normality. The ANOVA and the
Tukey’s test were applied to identify differences between treatment groups,
except in the analysis of TLR responses in PBMCs and NF-?B activity for
which the two-sided Dunnett’s test was applied. Statistical significance was
assigned at the value for p ? 0.05. For the analysis of Ab titers, statistical
significance was assigned at a value for p ? 0.05 and the difference be-
tween the groups was also ?2-fold.
MPL acts as a specific TLR4 agonist
LPS from the Gram-negative bacterium S. minnesota has been
reported to be a specific TLR4 agonist (10, 11). MPL, a detoxified
derivative of LPS, has also been shown to act through TLR4 (12),
although one study reported some TLR2 activity (13).
The contribution of TLR4 and TLR2 on MPL activity was there-
fore re-examined using clinical grade MPL alone or within AS04.
This examination was done using HEK cells transfected with plas-
mids encoding TLR4 and its coreceptors MD2 and CD14, or en-
coding TLR2 and TLR1, or encoding TLR2 and TLR6 (Fig. 1A).
Both MPL and AS04, but not aluminum hydroxide alone, induced
NF-?B activity in the tlr4/md2/cd14 transfectants, confirming that
MPL signals via TLR4. In contrast no substantial increases in
and symbols) CD14?monocytes in human PBMCs (n ? 4 donors). PBMCs in 5 ? 105cells/well (96-well plate) were stimulated as in Fig. 1B with medium
or a dose range of MPL or aluminum hydroxide at the indicated concentrations (in ?g/ml). B, The level of IL-1? detected in culture supernatants of human
PBMCs. PBMCs were stimulated with a dose range of MPL or aluminum hydroxide at the indicated concentrations (in ?g/ml) for 18 h without and with
caspase-1 inhibitor Y-VAd. Data represent geometric mean and the symbols represent the data points from each donor sample tested. C and D, Human
monocyte-derived DCs in 2 ? 105cells/well (96-well plate) were stimulated with the full vaccine (HPV-16 and HPV-18 L1 VLPs/AS04), Ags (HPV-16
or HPV-18 L1 VLP/aluminum hydroxide) or MPL at the concentrations indicated (in ?g/ml) (n ? 2 or n ? 4 mice). C, The levels of TNF-? and IL-6 after
18 h in the supernatant are shown. D, The proportion of CD1a?DCs expressing the costimulatory molecules CD83 (open symbols) and CD86 measured
by flow cytometry after 18 h. Data represent geometric means and the symbols represent the data points from each of donor samples tested. E, Human CD4?
T cells for n ? 6 donors in 2 ? 105cells/well (96-well plate) were stimulated for 24 h with medium alone, MPL (10 ?g/ml), anti-CD3 (1 ?g/ml), or MPL
(10 ?g/ml), and anti-CD3 (1 ?g/ml) and the proportions of CD69, IFN-?, and CD40L-expressing cells relative to the anti-CD3 condition is represented
as a box plot. The median of the n ? 6 donors is shown (horizontal line), and results are described by the 1st and 3rd quartiles with the lowest and highest
values shown by the whiskers.
AS04 stimulated human APCs but not T cells. A, The proportion of TNF-?-positive (open bars and symbols) and IL-6-positive (shaded bars
6189The Journal of Immunology
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NF-?B activity were detected in both tlr1/2 and tlr2/6 transfected
To evaluate TLR4 and TLR2 responses in a more relevant
model, PBMCs were stimulated for 6 h, and intracellular cytokines
were measured in activated CD14?monocytes. MPL induced the
production of IL-6 and TNF-? by monocytes and this response
was reduced when a TLR4 blocking Ab was added before MPL
(p ? 0.001, Fig. 1B). In contrast, an anti-TLR2 Ab had no effect
on MPL-induced monocyte activation (p ? 0.9514), whereas it
inhibited monocyte activation by a TLR2 agonist, peptidoglycan
(p ? 0.01). Altogether, these results indicated that MPL either
alone or adsorbed on aluminum hydroxide (as formulated in AS04)
acts as a TLR4 agonist that is in agreement with what was reported
by Tiberio et al. (14).
The stimulation of PBMCs with a dose range of AS04 or MPL
similarly induced CD14?monocytes to express IL-6 and TNF-?
(Fig. 2A) compared with stimulation with aluminum hydroxide
(p ? 0.0001 and p ? 0.001, respectively) in which IL-6?and
TNF-??monocytes remained at background levels. Therefore alu-
minum hydroxide in the AS04 preparation did not interfere with or
appear to enhance the TLR4-specific proinflammatory cytokine
production induced by MPL on human monocytes. However, there is
a potential for MPL and aluminum hydroxide to synergize in the
secretion of caspase-dependent cytokines, given their respective
influences on transcription and maturation of these cytokines. As
expected, the stimulation of PBMCs with a dose range of AS04 but
not with aluminum hydroxide alone induced the secretion of IL-1?
(p ? 0.001), which was inhibited by the addition of the caspase-1
specific inhibitor Y-VAd (Fig. 2B). A similar induction of IL-1?
was observed with MPL stimulation compared with aluminum hy-
droxide (p ? 0.01). This indicated that MPL was sufficient to
induce the caspase-1 dependent production of mature IL-1?.
Therefore, the combination of MPL and aluminum hydroxide did
not significantly synergize in the induction of cytokine secretion.
MPL and AS04 stimulate human DCs but not human T cells
DCs as well as T cells have been shown to express TLR4 (24, 25).
Human DC stimulation with a dose range of MPL or the vaccine
both significantly induced the production of TNF-? and IL-6 and
increased the surface expression of the costimulatory molecules
CD83 and CD86, compared with HPV-16 or HPV-18 L1 VLPs
adsorbed on aluminum hydroxide (Fig. 2D). Typically these re-
sponses were proportional to the concentration of MPL used.
Therefore MPL retained its ability to activate DCs when formu-
lated in the vaccine. Furthermore, MPL was the only component in
the vaccine that drove this response as the HPV Ags adsorbed on
aluminum hydroxide had no direct effect on these parameters of
Purified human CD4?T cells were stimulated with MPL in
the presence or absence of TCR engagement via anti-CD3 Abs
(Fig. 2E). Consistent with the study by Ismaili et al. (25),
CD40L was slightly increased in T cells but only upon con-
comitant TCR engagement. However, this difference was not
found to be significant when tested in six donors. In addition,
MPL did not significantly alter the expression of two other
markers of T cell activation, i.e., CD69 and IFN-?, without
consideration of anti-CD3 stimulation. Therefore MPL was able
to directly stimulate human monocytes and DCs, but unable to
significantly stimulate T cells.
Spatial and temporal colocalization of AS04 and Ags is required
for an optimal immune response
The superior adjuvant activity of AS04 over aluminum salt
alone has been previously demonstrated in mice following vac-
cination with HPV-16/18 L1 VLPs (22). To compare the adju-
vant activity of AS04 to MPL, C57BL/6 mice were injected
with HPV-16/18 L1 VLPs adjuvanted with AS04 or MPL alone,
twice at day 0 and day 14, and the humoral responses were
analyzed 14 days later. The addition of aluminum hydroxide to
MPL significantly enhanced the Ab response for both HPV-16
and HPV-18 L1 VLPs (from 7893 to 21614 EU/ml for HPV-16
and from 5758 to 25745 EU/ml for HPV-18; n ? 20, p ?
0.001), further demonstrating the added value of MPL and alu-
minum salt combination in the vaccine.
To identify the spatial and temporal requirements of AS04
adjuvant activity, different i.m. immunization protocols were
followed in C57BL/6 mice (Fig. 3). Effectively the vaccine dose
was split such that MPL per one-half dose of aluminum hy-
droxide was injected first and the Ags (HPV-16/18 L1 VLPs per
one-half dose of aluminum hydroxide) were then injected 1 or
24 h later either into the same limb site or into a separate site
on the contralateral limb. HPV-16/18 L1 VLPs, adjuvanted in
AS04 (the whole vaccine) or adjuvanted with only an equiva-
lent amount of aluminum hydroxide, were used as positive and
pared with aluminum hydroxide alone, when the Ags were injected in the
same site and within 24 h after the MPL/aluminum hydroxide injection.
Anti-HPV-16 L1 VLP geometric mean titers (A) and anti-HPV18 L1 VLP
geometric mean titers (B) were measured 14 days after i.m. immunization
in C57BL/6 mice. The quantity of the vaccine component (MPL; aluminum
hydroxide, and HPV-16 L1 VLP/HPV-18 L1 VLP) used in each immuni-
zation (first/second injection) was tabulated directly below the respective
results. Error bars represent ? 95% confidence intervals (n ? 18). ?, p ?
0.0001 for significant differences by Tukey’s test between the respective
geometric mean titers and the value observed after the injection of the Ags
adjuvanted with AS04 (VLPs/AS04).
AS04 induced high geometric mean titers (GMTs) com-
6190AS04 INDUCES A TRANSIENT LOCALIZED INNATE IMMUNE RESPONSE
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negative controls respectively. The impact of the adjuvant ac-
tivity on the humoral responses was assessed at 14 days after
the first injection.
The Ab response was not significantly different when the Ags
were injected 1 h after MPL and aluminum hydroxide at the same
site compared with the whole vaccine. However, when the Ags
were injected 24 h later than MPL and aluminum hydroxide, the
Ab response was significantly lower (?2.4-fold) than the response
observed with the vaccine. When the Ags were injected after MPL
and aluminum hydroxide but in the contralateral muscle, MPL and
aluminum hydroxide did not have any impact on the immuno-
genicity of the Ags and the Ab titers were not significantly
above the negative control (Ags adjuvanted with aluminum hy-
droxide alone). Therefore the superior adjuvant activity of
AS04 vs aluminum hydroxide was dependent on the Ags being
in the same location as AS04 within a period of 24 h.
The i.m. injection of MPL or AS04 leads to local NF-?B
A first evaluation of the vaccine localization in situ by immuno-
histochemistry indicated that MPL and the other vaccine compo-
nents were found at the site of injection for at least 24 h following
the vaccine injection (data not shown). Only a limited quantity of
Ag and no MPL could be detected in the lymph nodes, suggesting
that the vaccine components remain primarily local to the site of
Because MPL has been shown to induce NF-?B activation (13,
25), NF-?B activity was used as a readout for the functional re-
sponse to MPL to further characterize the localization of MPL.
This response was measured using a transgenic mouse model that
reveals NF-?B activity by expression of a luciferase reporter gene
The injection of AS04 into the right gastrocnemius muscle was
compared with the injection of PBS into the left gastrocnemius
muscle (Fig. 4A). AS04 resulted in higher NF-?B activity (p ?
0.01) in right hind limb compared with the contralateral limb at
5 h, but not at 22 h postinjection (Fig. 4A). This indicated that
AS04 transiently stimulated TLR4 signaling at the injection site.
Therefore, to examine the direct responses to MPL, NF-?B ac-
tivity was also measured in surgically exposed lymph nodes at 5 h
postinjection. Compared with PBS, the injection of MPL and
AS04 resulted in a 4.7-fold (p ? 0.001) and 2.8-fold (p ? 0.05)
higher NF-?B activity, respectively, in the hind leg draining iliac
lymph node (Fig. 4B). In the right inguinal lymph node, the in-
jection of MPL also resulted in 4.0-fold higher (p ? 0.05) NF-?B
activity (Fig. 4B), in contrast to the left inguinal lymph node that
does not drain the injection site.
As a measure of systemic responses to MPL, NF-?B activity
was examined in spleen homogenates (Fig. 4C). MPL i.v. injec-
tion, but not MPL or AS04 i.m. injection, resulted in NF-?B ac-
tivity in the spleen (p ? 0.001). Therefore, following AS04 i.m.
injection, the direct response to MPL was restricted to the injection
site and draining lymph node.
Local cytokine induction by MPL and AS04
NF-?B activity is associated with an innate immune response that
results in the induction of cytokines. Proinflammatory cytokines,
secreted by resident and recruited cells, directly stimulate cells that
then present the Ag in the draining lymph node. Chemokines play
a key role in inducing the recruitment of various immune cells at
the injection site. The contribution of MPL and the other vaccine
components to the innate immune response at the injection site was
therefore further examined by measuring the levels of proinflam-
matory cytokines (IL-6, TNF-?, and IFN-?) and chemokines
(CCL2/MCP-1 and CCL3/MIP-1?) in homogenates prepared from
injected muscle at 3, 6, and 24 h and 7 days postinjection (Fig. 5A).
PBS was injected as a baseline negative control. However, IFN-?
was not induced by any of the vaccine component formulations.
The injection of the MPL containing formulation (but not Ags
and aluminum hydroxide, aluminum hydroxide alone, or Ags
alone) resulted in ?10 fold increases in the levels of IL-6, TNF-?,
CCL2, and CCL3 compared with the PBS injection (Fig. 5A).
These results indicated that MPL, and not the Ags or aluminum
hydroxide, was the principal component in the vaccine that medi-
ated the early cytokine response.
NF-?B-luciferase transgenic mouse model. A, NF-?B activity (n ? 6; measured by live imaging) at 0, 5, and 22 h after injections of AS04 in the right
gastrocnemius and PBS in the left gastrocnemius. The NF-?B activity in the right hind limb is normalized to the NF-?B activity of the respective left limb.
Geometric mean (F) and data points from each of the mice tested (E) are shown. Significant difference is indicated (?) by Dunnett’s test relative to the 0 h time
The measurements were taken 5 h after the injection of PBS (control), MPL or AS04 into the right gastrocnemius and PBS into the left gastrocnemius, or after
the i.v. injection of MPL. All values were normalized to the respective measurement with PBS injection. Significant difference is indicated (?) by Dunnett’s test
with respect to the PBS injections. Data represent geometric mean and symbols represent the data points from each of the mice tested.
AS04 i.m. injection stimulated NF-?B activity only at the injection site. NF-?B activity was revealed by measuring luminescence using the
6191The Journal of Immunology
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The production of cytokines was transient as the maximal levels
were detected between 3 and 24 h following injection. Moreover,
for a given cytokine, the peak of the MPL response was generally
preceding those of the vaccine or MPL and aluminum hydroxide.
Also at 24 h and 7 days, the responses induced by AS04 were
typically higher than the ones induced by MPL. These results
showed that aluminum hydroxide in AS04 prolonged the MPL-
mediated cytokine response, especially between 6 and 24 h.
Maximal levels CCL2, CCL3, and TNF-? were detected at 6
or 24 h after vaccine and after MPL and aluminum hydroxide
injections, later than the maximal levels of IL-6, which was de-
tected at 3 or 6 h. This suggested that the delayed dynamic of the
CCL2, CCL3, and TNF-? responses reflect different regulatory
pathways being activated or the additional contribution of newly
recruited cells to their expression levels mainly at 24 h. The in-
jection of aluminum hydroxide resulted in ?5-fold increase in the
levels of CCL2, detectable only 24 h after injection (Fig. 5A). The
levels of the other cytokines were not increased to a great extent at
any of the time points tested. So although aluminum hydroxide had
a minor impact on some cytokine production, MPL was the main
driver of the cytokine response observed in AS04.
In the serum, the i.m. injection of the vaccine resulted in a ?5-
fold increase in the levels of IL-6, but not of CCL2 compared with
PBS (and measured between 1 h to 3 days after injection) (Fig.
5B). However, these cytokine levels were typically ?10-fold
lower than those found in the muscle homogenates, and were sub-
stantially much lower than those found with a systemic injection of
1 ?g of MPL. The irrelevance of serum cytokines for the induction
of an adaptive response was further demonstrated by evaluating
the i.m. injection of one-fifth the dose of the vaccine (10 ?l). The
injection of 10 ?l of the vaccine resulted in negligible or no
changes to serum cytokine levels compared with the PBS injection
(Fig. 5B). Yet injections with 10-?l doses of the vaccine gave a
similar Ab response compared with injections with the full dose
(50 ?l) of the vaccine (Fig. 5C).
The substantially lower cytokine response detected in serum, as
compared with the muscle, following i.m. injection of MPL and
aluminum hydroxide or vaccine further indicated that the innate
response remained local to the injection site. Furthermore, the ab-
sence of NF-?B activity in spleen with AS04 injections, also sup-
port the assumption that cytokines in the serum most likely dis-
seminated from the local injection site rather than from de novo
production in distant organs. The cytokine responses in both mus-
cle homogenates and in serum samples were similar in BALB/c
mice compared with C57BL/6 mice, suggesting that these re-
sponses are not strain-dependent (see supplemental Fig. 1).7The
results therefore indicated that the temporal and spatial constraints
on AS04 superior adjuvant activity coincided with the transient
induction of an innate immune response by AS04 acting locally at
the injection site.
MPL and AS04 stimulate the infiltration of DCs and monocytes
into the draining lymph node
DCs and monocytes, once activated at the site of injection by
the local innate response, can migrate to the draining lymph
node to provide essential information, including the antigenic
determinants, to T and B cells for the generation of an adaptive
response (26, 27). DC numbers and activation status were first
assessed by flow cytometry in pooled draining iliac lymph
7The online version of this article contains supplemental material.
lations injected into muscle of C57BL/6
mice stimulated transient cytokine pro-
duction at the site of injection. A, IL-6,
centrations measured in homogenized
cle preparations taken 3, 6, and 24 h or
7 days after i.m. injection of the vaccine
(MPL/Alum/VLP; 5/50/4 ?g), MPL/
aluminum hydroxide (MPL/Alum; 5/25
?g), Ags/aluminum hydroxide (VLP/
Alum; 4/25 ?g), aluminum hydroxide
(Alum; 25 ?g), or Ags alone (VLP; 4
?g), MPL (5 ?g) or PBS. B, IL-6 and
CCL2 concentrations measured in se-
rum samples (n ? 6) taken 1, 3, 6, and
24 h or 3 days after an i.m. injection of
50 or 10 ?l of the vaccine (VLP/AS04),
PBS, or after an i.v. injection of a 5-fold
lower dose of MPL. These data are rep-
resentative of at least two experiments.
C, Anti-HPV16 L1 VLP geometric
mean titers (GMTs) and anti-HPV18 L1
VLP geometric mean titers (n ? 12
mice) measured 14 days after two suc-
cessive i.m. injections (day 0 and day
14) of 50- or 10-?l vaccine.
6192 AS04 INDUCES A TRANSIENT LOCALIZED INNATE IMMUNE RESPONSE
by guest on June 13, 2013
nodes taken from mice at 6, 24, or 72 h following i.m. injection
with the different components of the vaccine (Fig. 6A). At 6 h,
the DC levels were similar for all of the formulations injected
including PBS (data not shown). At 24 and 72 h, the injection
of the MPL-containing formulation induced 3.4- to 13.5-fold
higher numbers of DCs compared with the aluminum hydrox-
ide-only formulations or PBS (p ? 0.001) (Fig. 6A). A higher
number of DCs expressing high levels of MHC class II were
also observed both at 24 and 72 h with these injections (p ?
0.001) (see supplemental Fig. 2). Such MHC class IIhighcells
are indicative of recently activated DCs that have migrated from
the site of injection into the draining lymph node (28, 29).
The increased expression of costimulatory molecules CD40 and
CD86 provides essential signals to naive T cells to initiate an op-
timal adaptive response. The levels of CD40 and CD86 were quan-
tified by flow cytometry on DCs (Fig. 6A). At 24 and 72 h, only the
injection of MPL-containing formulations induced higher levels of
CD40 and CD86 in the DCs as compared with the injection of PBS
(p ? 0.0001).
Monocytes have the capacity to differentiate into DCs (27, 30)
and such a differentiated population can be detected by the expres-
sion of the cell surface markers Ly6C, CD11b, and F4/80 (31, 32).
The levels of these triple positive cells were also quantified by flow
cytometry (Fig. 6A). At 24 and 72 h, the injection of MPL-con-
taining formulations induced 3.7- to 26-fold higher numbers of
monocytes compared with the aluminum hydroxide-only formula-
tions or PBS (p ? 0.01); and compared with PBS, also induced
higher levels of CD40 (p ? 0.05), but not of CD86.
Confirming the local induction of immunity by the vaccine or its
components, the levels of CD40 or CD86 in the DCs isolated from
nondraining axillary lymph nodes were not significantly increased
at 24 h after injection (see supplemental Fig. 2A).7Similarly, the
DCs of the spleen were also unaffected at 6 h postinjection (the
anticipated time point to observe a systemic response) (see sup-
plemental Fig. 2A).7The spleen and axillary lymph node DCs
were capable of responding because the i.v. injection of MPL com-
pared with PBS resulted in a ?2-fold induction of CD40 in the
axillary lymph node DCs and ?2-fold induction of CD86 in spleen
DCs (see supplemental Fig. 2B).
Ag-loaded cells were tracked in the draining lymph nodes using
fluorescent-labeled OVA in combination with various formula-
tions. Results generated with OVA were comparable to the ones
generated previously with L1 VLP Ags (Fig. 6A). A higher DC and
monocyte count per lymph node was observed at 24 h postimmu-
nization (p ? 0.05) (Fig. 6B). Hence there was no evidence that
the MPL-induced innate response was affected by using a different
lated increased DC and monocyte numbers in draining
iliac lymph nodes. Cells from pooled iliac lymph nodes
were analyzed by flow cytometry. A, C57BL/6 mice
were immunized with the vaccine (MPL/Alum/VLP;
5/50/4 ?g), MPL/aluminum hydroxide (MPL/Alum;
5/25 ?g), Ags/aluminum hydroxide (VLP/Alum; 4/25
?g), aluminum hydroxide (Alum; 25 ?g), Ags alone
(VLP; 4 ?g), MPL (5 ?g) or PBS (n ? 3 pools in which
each data point is derived from pooled sample from n ?
6 mice). The number of DCs (gated as CD11c?MHC
class II-positive) per lymph node or monocytes (gated
as Ly6C?F4/80?and CD11b?) per lymph node at 24
or 72 h following i.m. injection. The mean fluorescent
intensity (MFI) of CD40 or CD86 expression in the total
population of DCs or monocytes at 24 or 72 h. B,
C57BL/6 mice were immunized by i.m. injection of
fluo-OVA (5 ?g) as the Ag, in combination with MPL
(5 ?g) and aluminum hydroxide (Alum; 50 ?g) as in-
dicated (n ? 4 pools in which each data point is derived
from pooled sample from 6 mice). The number of total
and OVA-positive DCs and monocytes per iliac lymph
node at 24 h are shown. The mean fluorescent intensity
(MFI) of CD40 or CD86 expression in the OVA-posi-
tive population of DCs or monocytes. Data represent
geometric mean and symbols represent the data points
from each of the pooled samples tested.
MPL-containing formulations stimu-
6193 The Journal of Immunology
by guest on June 13, 2013
Ag. Moreover, higher OVA-positive DCs and monocyte numbers
per lymph node (p ? 0.05), expressing higher levels of CD40 and
CD86 (p ? 0.01) were observed with the MPL-containing formu-
lations. These results indicated that MPL in AS04 stimulated an
increased migration of Ag-loaded and activated DCs and mono-
cytes to the draining lymph nodes at 24 h.
The direct impact of the adjuvants on DC activation and Ag
uptake was further analyzed using BMDCs stimulated in vitro with
the different adjuvant formulations in combination with fluores-
cently labeled OVA. In a dose-dependent manner, aluminum hy-
droxide, MPL, or the combination of aluminum hydroxide and
MPL similarly enhanced Ag uptake compared with BMDCs
treated without adjuvant (Fig. 7A). However, only the MPL-con-
taining adjuvants induced a 2-fold induction of CD86?BMDCs.
Hence the effects on in vitro Ag uptake and DC activation are also
consistent with what was observed in vivo with the DCs in drain-
ing lymph nodes.
To investigate whether increased Ag uptake and costimulatory
molecule expression induced by AS04 would translate into the
activation of Ag-specific T cells, OVA-specific CFSE-labeled-
DO11.10 CD4?T cells were incubated for 72 h with BMDCs
pretreated with OVA alone or in combination with MPL or alu-
minum hydroxide. T cell proliferation was evaluated by measuring
the decrease in CFSE labeling in proliferating cells. Only the
BDMCs stimulated with MPL-containing adjuvants induced the
specific proliferation of DO11.10 T cells (Fig. 7B). These results
show that MPL in AS04, in contrast to aluminum hydroxide, better
stimulated DCs for Ag presentation to specific T cells. Similar
results were observed with the examination of BDMCs from another
strain of mouse, suggesting again that this observation is not strain-
dependent (see supplemental Fig. 3).
The same functional capacity of DCs was identified in vivo in
draining lymph nodes 24 h after immunization. CD11c?DCs from
mice injected with OVA alone, or in various formulations, were
purified from pooled draining lymph nodes and incubated with
CFSE-labeled D011.10 T cells. DCs purified from mice injected
with OVA/AS04 or OVA/MPL stimulated more proliferation in
CD4?T cells compared with DCs from mice injected with OVA
and aluminum hydroxide or OVA without adjuvant (Fig. 7C). The
Ag-specific T cell response after vaccination was examined in
splenocyte cultures from vaccinated BALB/c mice. Four mice per
treatment group were i.m. injected twice 14 and 35 days previ-
ously, with HPV 16/18 L1 VLP Ags alone or in combinations with
MPL and aluminum hydroxide. IL-6, IFN-?, and IL-5 production
in the culture supernatants were used as a measure of T cell acti-
vation following 48 h in vitro stimulation with HPV-16 VLP or
HPV-18 L1 VLP Ags (Fig. 7D).
Levels of IL-2, a marker of pan-T cell proliferation, were higher
in the aluminum hydroxide or AS04 immunized mice groups com-
pared with the Ag only group (p ? 0.0001). IL-2 levels were also
higher with AS04 compared with aluminum hydroxide (p ? 0.05).
Levels of IFN-?, a marker of Th1 bias, were higher with AS04
compared with aluminum hydroxide (p ? 0.0001). Conversely,
in 96-well plate for 6 h with a dose range of fluo-OVA (1 and 10 ?g/ml) alone or in combination with MPL (1 and 10 ?g/ml, respectively) or aluminum
hydroxide (Alum, 10 and 100 ?g/ml, respectively). Cells were analyzed by flow cytometry, and mean fluorescent intensity (MFI) of fluo-OVA-positive
and CD86 in CD11c?BMDC is shown. Data represent mean (n ? 3 mice) and error bars represent SD. B, BALB/c BMDCs were stimulated at 1 ? 106
cells/ml in 24-well plate for 6 h with a dose range of fluo-OVA (1 and 10 ?g/ml) alone or in combination with MPL (1 and 10 ?g/ml, respectively) or
aluminum hydroxide (Alum, 10 and 100 ?g/ml, respectively). After an extensive wash, BMDC were cocultured for 3 days with OVA-specific TCR
transgenic DO11.10 T cells with the ratio of 1:10 for BDMC to T cells (105T cells/well in a 96-well plate). Representative histograms showing CFSE profile
of CD4?KJ1.26?DO11.10 T cells are shown. Mean percentage ? SD (for n ? 3 mice) of CFSElowCD4?T cells, indicative of proliferation are also
shown. C, CD11c?cells were isolated by magnetic-positive selection from pooled draining iliac lymph nodes of mice i.m. injected 24 h previously with
OVA (5 ?g) alone or in combination with MPL (5 ?g) and aluminum hydroxide (Alum; 50 ?g) as indicated (pool of 24 mice per immunization). Purified
DCs were then coincubated for 3 days with DO11.10 T cells with a ratio of 1:2, 1:5, 1:10, 1:20, or 1:40 DCs to T cells as indicated by differently shaded
bars (105DO11.10 T cells/well in a 96-well plate). Proliferation was measured as the percentage of CFSElowT cells as described in B. Data represent mean
(n ? 3 cell cultures) and error bar represents SD. D, Splenocytes were isolated from mice i.m. injected 14 and 35 days previously with HPV-16 and HPV-18
VLP Ags (2 ?g) alone or in combinations with MPL (5 ?g) and aluminum hydroxide (Alum; 50 ?g) as indicated. The cells were then stimulated with
HPV-16 and HPV-18 L1 VLPs for 48 h (5 ? 106cells/well; 24-well plate) and IL-6, IFN-?, and IL-5 concentrations were measured in the supernatant
(n ? 4 pools in which each data point is derived from one mouse). Data represent geometric mean and symbols represent the data points from each of the
splenocyte culture samples tested.
MPL- and AS04-mediated activation of DCs is associated with T cell activation. A, BALB/c BMDCs were stimulated at 1 ? 106cells/ml
6194AS04 INDUCES A TRANSIENT LOCALIZED INNATE IMMUNE RESPONSE
by guest on June 13, 2013
the levels of IL-5, a marker of Th2 bias, were higher with alumi-
num salt and with the Ag alone compared with AS04 (p ? 0.01).
These results indicated that AS04 was a better inducer of CD4?T
cell amplification and differentiation compared with aluminum hy-
droxide alone, and promoted a Th1 bias characteristic of a TLR4
Altogether, these results demonstrated that within AS04, MPL
was the principal driver of APC increased numbers and activation,
and that these responses were restricted to the draining lymph
node. At 24 h after injection, the results also demonstrated that
MPL adsorbed on aluminum hydroxide induced a substantial wave
of migrating Ag-loaded APCs to enter the draining lymph node
with the enhanced capacity to directly stimulate T cells.
AS04 was designed to use a well-defined immunostimulant, MPL,
in combination with aluminum salt (33). The benefit of this ap-
proach has been shown by better adaptive immune responses in
mice, monkeys, and humans to immunization with vaccines con-
taining AS04 compared with the same Ags formulated with alu-
minum hydroxide alone (22). In this study, by using multiple ap-
proaches in vivo and in vitro, we show that the addition of MPL to
aluminum hydroxide led to the rapid and spatially localized
activation of an innate response, which could explain how a
robust adaptive immune response is achieved in AS04-adju-
Crucial role of MPL in AS04 for the stimulation of an innate
Our results show that MPL was responsible for the transient in-
duction of cytokines and NF-?B activity observed within the first
day following the injection of AS04, consistent with MPL acting
as a TLR4 agonist (25, 34–36). MPL was also responsible for the
infiltration and activation of DCs and monocytes in the draining
lymph nodes by 24 h. This report is the first to our knowledge
describing the local effect of MPL after i.m. immunization, the
most common route used for vaccination in humans. Early induc-
tion in the muscle of NF-?B 5 h after injection was indicative of
MPL directly stimulating TLR4-expressing cells. Various cell
types in the muscle can express TLR4, and therefore could have
been responsible for the early NF-?B activity and cytokine secre-
tion (24, 37, 38), although infiltrating cells were likely to have also
contributed at later stages. NF-?B induction was also observed in
the draining lymph node at 5 h. This finding could have been due
to low levels of MPL draining to the local lymph node and directly
activating resident cells. Alternatively, cytokines produced in the
muscle could enter the draining lymph node and in turn induced
NF-?B activity. Activated cells directly migrating from the blood
to the draining lymph nodes may have also contributed further to
cytokine-induced NF-?B activity. Importantly, the spatial confine-
ment of NF-?B activity to the injection site and draining lymph
node was demonstrative of a localized rather than a systemic im-
mune response to AS04 injection.
Aluminum hydroxide made little contribution to the early innate
response stimulated by AS04. In addition, there was no evidence
that aluminum hydroxide acted synergistically with MPL to en-
hance the magnitude of cytokine production (in vitro or in vivo) or
to enhance the infiltration of APC in the draining lymph nodes 24 h
after injection. Neither did aluminum hydroxide alter substantially
the type of cytokines and recruited cells induced by MPL. How-
ever, aluminum hydroxide did prolong the cytokine responses at
the injection site.
Although MPL and aluminum hydroxide could potentially co-
operate in increasing IL-1? secretion (19, 20), in this study MPL
stimulated IL-1? secretion independently of aluminum hydroxide
in vitro. This observation is in line with a recent report by Maelfait
et al. (39), which showed that LPS can induce the cleavage of
pro-IL-1?. However, the authors demonstrated that this occurred
via the activation of caspase-8 and not by caspase-1 as reported.
Hence further work is required to evaluate the contribution of each
of these caspases in the control of IL-1? secretion induced by
In contrast to the present study, Kool et al. (30) showed that
aluminum salt induced a strong and rapid inflammatory response
using an i.p. route of administration. Even though a higher dose of
aluminum salt was used, the discrepancy highlights the potential
dependence of the adjuvant response on the route of immunization.
The results in this present study are more consistent with a recent
microarray analysis reporting only a few genes being altered in
muscle upon aluminum injection compared with other adjuvants
(40). But in common with Kool et al. (30), aluminum hydroxide
stimulated Ag uptake and induced CCL2 production. These effects
of aluminum salt have been linked to monocyte differentiation and
migration to draining lymph nodes.
Altogether, this study suggested that the presence of aluminum
hydroxide, through its recently described mode of action and its
impact on cytokine response, has an overall beneficial impact on
MPL response. Importantly, aluminum hydroxide also plays an
important role in the physical stabilization of VLP Ags and MPL
in the vaccine formulation, as well as spatially confining the Ag
and MPL to the injection site.
HPV-16 and HPV-18 L1 VLPs appeared not to contribute to the
early vaccine innate response compared with AS04 as shown by
their inability to induce cytokine production and APC activation in
vivo and in vitro. HPV L1 VLPs have been reported elsewhere to
induce cytokine response in DCs, monocytes, and macrophages
(41–43), possibly in a TLR-dependent fashion (44, 45). Abs
against HPV L1 VLPs were found to be reduced in TLR4-deficient
mice by Yang et al. (44) but not by others (46). An explanation for
these discrepancies could come from the differences in the HPV L1
VLP preparation or in the experimental models used. In particular,
the adsorption of HPV L1 VLPs on aluminum salt may explain
their inability to stimulate innate cells.
How does AS04 enhance Ab response compared with aluminum
The adjuvant activity of AS04 compared with aluminum salt has
been shown to induce higher levels of HPV L1 VLP-specific Abs
and memory B cells in humans (22), demonstrating that AS04 is
able to promote a B cell response either directly or indirectly.
Recent studies clearly indicate that, contrary to murine B cells
(47), human purified B cells do not express TLR4 and are not able
to respond to LPS stimulation, even during concomitant BCR stim-
ulation. Therefore this evidence does not support a role for AS04
in directly activating B cells (48–50). Similarly it is unlikely that
T cells are directly activated by AS04. CD4 T cell effector func-
tions (as measured by CD40L and IFN-?) were not significantly
induced or enhanced by MPL in this present study. In some stud-
ies, T cell responses to TLR4 agonists have been observed, such as
increased adherence and Ca2?mobilization, but these measures
are not necessarily indicators of Ag-independent T cell activation
(25, 51, 52). Therefore, it is likely that the superior adjuvant ac-
tivity of AS04 resulted from APC-mediated activation of T cells
and subsequent B cell activation. This is supported by the rapid
appearance of Ag-loaded and activated APCs in the draining
lymph nodes. In such conditions, efficient Ag uptake, Ag process-
ing, and APC-mediated presentation to T cells would be occurring
at a time when the local Ag concentration is at its peak.
6195The Journal of Immunology
by guest on June 13, 2013
The cytokines induced at the injection site are functionally rel-
evant to the cellular events detected in the draining lymph nodes.
Indeed, chemokines such as CCL2 and CCL3 are known to pro-
mote the recruitment of monocytes and immature DCs (53). In
particular, CCR2, the receptor for CCL2 has been shown in many
models of infectious diseases and vaccination to be crucial for the
mobilization of monocytes and the induction of an immune re-
sponse (54–56). Differentiated monocytes and DCs are key medi-
ators for the induction of T and B cell responses, as they both can
take up Ags and migrate to the draining lymph nodes (55). In this
study, the combination of MPL and aluminum hydroxide led to an
optimal APC recruitment and activation in the draining lymph
node, which could be due in part to aluminum hydroxide prolong-
ing the cytokine response to MPL. The direct activation of DCs by
MPL may also be crucial for a sustained Ab response. Indeed, it
has been argued that the direct activation of DCs by TLR agonists
enhance their ability to promote Ag-specific immunity rather than
if they were activated by cytokines alone (26). IL-6 has been
shown to play a role in the Ab response induced by an MPL-based
vaccine (57) and this cytokine has been shown to promote Ab
response by stimulating Th cells specialized in providing help to B
This study has demonstrated that MPL in the vaccine can en-
hance the quality of the Ag specific T cell response. This response
is associated with the rapid migration of Ag loaded and activated
DCs to the draining lymph nodes. The elevated expression of
CD40/86 in DCs is known to be critical for the Ag-specific acti-
vation of Th cells (59) and the optimal activation of APCs favors
the local induction of Th cells expressing TCRs with the highest
affinity to antigenic peptides (60). An MPL-based adjuvant has
been found to specifically activate local follicular Th cells that are
thought to be directly associated with the generation of B cell
memory (61, 62). An appealing hypothesis is, therefore, that the
superior adjuvant activity of AS04 is due to the generation in the
draining lymph node of follicular Th cells with a high affinity TCR
repertoire, but this remains to be determined.
Localized and transient activity of AS04 supports the favorable
clinical safety profile
The localized and transient nature of the innate immune response
supports the favorable safety profile for the AS04 formulated vac-
cines observed in humans (63, 64). In particular, proinflammatory
cytokines were transiently induced and limited to the injection site,
and were absent or at very low levels in the serum. Moreover,
factors implicated in chronic inflammatory and autoimmune dis-
ease (65, 66) such as IFN-? induction, direct and relevant T cell
activation, and sustained high cytokine levels were not observed.
In conclusion, this study supports a model for the mechanism of
action of AS04 that can explain its advantageous adjuvant and
safety properties in human vaccines. In this model, MPL in AS04
plays a central role and demonstrates that modulation of innate
response by well-characterized agonists such as certain TLR ago-
nists in an appropriate formulation can provide an effective and
safe way to enhance vaccine responses in humans.
We thank Nabila Amanchar, Aurelie Chalon, Bernard Hoyois, Hajar Larbi,
Michel Fochesato, Laurent Renquin, Annie Leroy, My Hanh Le Thi,
Stephanie Quique, Luc Scholliers, Christel Verachtert, and Carsten Walter
for excellent technical support. We thank Dr. Matthew Morgan (4Clinics)
for writing and editorial assistance, and Dr. Ulrike Krause and Dr. Luise
Kalbe for editorial assistance. We thank Dr. Georges Carletti and Florence
Nozay for the statistical analysis. We are also grateful to Dr. Michel Gold-
man and Dr. Oberdan Leo for critical review of the manuscript.
Arnaud Didierlaurent, Sandra Morel, Laurence Lockman, Sandra Giannini,
Michel Bisteau, Nathalie Vanderheyde, Daniel Larocque, Marcelle Van
Mechelen, and Nathalie Garc ¸on are employees of GlaxoSmithKline Bio-
logicals. Francesca Schiavetti is a former employee of GSK Biologicals.
The remaining authors have no financial conflict of interest. Cervarix and
Fendrix are trademarks of the GlaxoSmithKline group of companies.
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