AS04 System

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of June 22, 2011This information is current as

http://www.jimmunol.org/content/183/10/6186doi:10.4049/jimmunol.0901474

October 2009;2009;183;6186-6197; Prepublished online 28J Immunol

Nathalie GaronSchiavetti, Daniel Larocque, Marcelle Van Mechelen andKielland, Olivier Vosters, Nathalie Vanderheyde, FrancescaSandra L. Giannini, Michel Bisteau, Harald Carlsen, AndersArnaud M. Didierlaurent, Sandra Morel, Laurence Lockman,Leading to Enhanced Adaptive ImmunityTransient Localized Innate Immune ResponseAgonist-Based Adjuvant System, Induces aAS04, an Aluminum Salt- and TLR4

DataSupplementary

74.DC1.htmlhttp://www.jimmunol.org/content/suppl/2009/10/28/jimmunol.09014

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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 Garcon*

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

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. The Journal of Immunology, 2009, 183: 61866197.

The development of new vaccines has highlighted the need

for new strategies to enhance and tailor the immune response

for effective and long-lasting protection. In particular, vac-

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)6 consisting of MPL (3-O-desacyl-

4-monophosphoryl lipid A) adsorbed onto a particulate form of

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) (35) 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 (1214), 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 responsetoward 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; Institutefor Medical Immunology, Universite Libre de Bruxelles, Charleroi, Belgium; andGlaxoSmithKline Biologicals, Laval, Canada

Received for publication May 11, 2009. Accepted for publication September16, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by GlaxoSmithKline Biologicals (to H.C. and A.K.).

2 A.D. and S.M. contributed equally to this work.

3 Address correspondence and reprint requests to Dr. Arnaud Didierlaurent, Glaxo-SmithKline Biologicals, Rue de lInstitut 98, 1330 Rixensart, Belgium. E-mailaddress: [email protected]

4 Current address: Institut de Recherche Interdisciplinaire en Biologie Humaine etMoleculaire, Universite Libre de Bruxelles, Brussels, Belgium.

5 Current address: Novartis Vaccines, Sienna, Italy.

6 Abbreviations 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

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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 TLR4signaling. 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

Vaccine formulationA 50-l dose of vaccine contained 5 g of clinical grade MPL (purifiedfrom S. minnesota), 2 g each of HPV-16 and HPV-18 L1 VLPs Ags (forpreparation of the Ags, see Ref. 22) and 50 g of aluminum hydroxide(Brenntag). The 50-l vaccine dose used in this study represents one-tenthof 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)

cells

Transfected HEK 293 cells (InvivoGen) with the expression vectors en-coding 1) TLR4, MD-2, and CD14, 2) TLR1 and TLR2, or 3) TLR2 andTLR6 were further stably transfected with the NF-B reporter vectorpNifty-2 secreted alkaline phosphatase (Invivogen). Cells were cloned by

zeomycin (50 g/ml) with further selection on the basis of appropriateTLR-stimulated expression of secreted alkaline phosphatase. Transfectedcell lines were plated at 2 105 cells per well in a 24-well plate culturedin 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 hin FCS-free medium containing MPL and aluminum hydroxide as indicatedor with LPS (0.1 g/ml, from S. minnesota; InvivoGen), PAM2CSK4(S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-cysteinyl-(S)-seryl-(S)-lysyl-(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 Blueassay (InvivoGen).

Preparation and in vitro stimulation of human cells

PBMC were isolated from the buffy coat fraction obtained from routineblood donor donations. Mononuclear cells were obtained by centrifugation

over Ficoll-Hypaque gradients. PBMCs were cultured in complete mediumcontaining RPMI 1640 supplemented with 50 M 2-ME, 1 mM sodiumpyruvate, 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine,

1% nonessential amino acids (Life Technologies), and 5% FCS (PAALaboratories). To measure intracellular cytokine production in monocytes,PBMCs at indicated concentrations were stimulated in triplicate in 96-wellplates at 37C 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. Aftersurface staining with CD14 Abs, cells were labeled intracellularly for IL-6and TNF- and analyzed by flow cytometry. In some experiments, PBMCswere preincubated for 1 h at 37C 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, 1 106 PBMCs were stimulated in 96-well plates for 18 h andsupernatants were collected in triplicates. The caspase-1 inhibitor Y-VAD (Calbochem) was used at 10 g/ml. Human IL-1 was measured bycytokine bead array (CBA; BD Biosciences). Human DCs were preparedfrom PBMC-derived monocytes treated with GM-CSF (100 ng/ml; R&DSystems) and IL-4 (100 U/ml; PeproTech) for 6 days. Cells were resus-pended in complete medium at 1 106 cells/ml and stimulated in 96-wellplates 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 byflow cytometry. CD4 T cells (from six donors) were purified from freshPBMC using a MACS column and a negative selection CD4 T cell iso-lation kit (Miltenyi Biotec). The whole isolation procedure was conductedat 4C and purity of95% was confirmed by flow cytometry. PurifiedCD4 T cells were cultured at 2 105 cells/200 l of RPMI 1640 with10% 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 wereanalyzed by flow cytometry after intracellular staining.

Mice immunization

All experiments and assays were performed in accordance with the localnational 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 tibialisanterior 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 measuredby 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

reporter mouse

NF-B activation was monitored in transgenic NF-B-luciferase mice inwhich expression of luciferase is regulated by three NF-B response ele-ments. In vivo imaging of NF-B activity was performed noninvasively orin mice with surgically exposed lymph nodes following i.p. injection ofD-luciferin (120 mg/kg; Biosynth). Time from D-luciferin injection to im-age acquisition was 10 and 25 min, respectively. Acquisition time wastypically 12 min using the IVIS100 Imaging System (Xenogen), com-

posed of a light-sealed imaging chamber fitted with a heating platform(37C) and cooled CCD camera (105C). Image acquisition and analysiswere done with the accompanying IVIS100 software. During the imagingperiod, mice were anesthetized with 2.5% isoflurane. Immediately after invivo imaging, mice were killed by cervical dislocation and the dissectedspleen specimens were snap-frozen in liquid nitrogen. Individual frozenorgans were homogenized in reporter lysis buffer (Promega) and cleared bycentrifugation. Luciferase activity was quantified (conventional luminom-eter; Turner Instruments) in the supernatant by adding luciferin, ATP, andmagnesium, following the manufacturers 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 eachpooled muscle sample, injected gastrocnemius muscles were pooled andhomogenized using an Ultraturax (VWR) in 3 ml of PBS plus anti-protease

inhibitor mixture (Sigma-Aldrich). The homogenates were then cleared bycentrifugation at 14,000 rpm for 10 min, and supernatants were stored at70C before analysis. Serum samples were measured from individual

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mice. Cytokine levels were measured according to the manufacturers 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 iliaclymph nodes were prepared using a Potter Homogenizer in medium (RPMI1640, 10% FCS, 100 U/ml of penicillin, 100 g/ml streptomycin 2 mML-glutamine, 50 M 2-ME, 1 mM sodium pyruvate) and passed through a100-m filter. Cells were then washed and counted using a Multisizer(Beckman Coulter).

Measurement of OVA uptake

Bone marrow DCs (BMDCs) were prepared from bone marrow cellstreated with mouse GM-CSF (10 ng/ml; R&D Systems) for 6 days.BMDCs were resuspended in complete medium at 1 106 cells/ml andincubated in 96-well plates in triplicate with Fluo-OVA-containing formu-lations at the indicated concentrations. Five hours later, the cells werewashed extensively and the level of Fluo-OVA uptake was analyzed byflow cytometry. In vivo, mice (n 4; 4 pools of 6 mice/group) wereimmunized i.m. with Fluo-OVA-containing formulations as indicated. Af-ter 24 h, cells were prepared from pooled iliac lymph nodes and analyzedby flow cytometry.

Measurement of Ag presentation to OVA-specific T cells

OVA-specific CD4 T cells were purified from pooled spleen and lymphnodes of OVA-TCR transgenic DO11.10 and OT-II mice using a CD4 Tcell negative selection kit (Dynal Biotech) according to the manufacturersrecommendations. Purified CD4 T cells were then stained with CFSE.Briefly, 1 107 T cells/ml were incubated with 5 M CFSE in RPMI 1640for 10 min at 37C. Cold RPMI 1640 plus 5% FCS was added to the cellsto 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 1 106 cells/ml were incubated in complete mediumwith OVA-containing formulations at the indicated concentrations for 6 h

in 24-well plates and washed extensively. Lymph node CD11cDCs werepurified using CD11c selection kit (Miltenyi Biotec) from pooled iliaclymph nodes from mice that were immunized 1 day before with OVA-

containing formulations (n

24 mice per vaccination). Treated BMDCs orpurified lymph node DCs were incubated with 1 105 CFSE-labeledOVA-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 wasassessed by flow cytometry.

Flow cytometry

Cells were resuspended in PBS, 1% FCS, and 1 mM EDTA and stainedwith the Abs described. To measure activation of human DCs, cellswere stained using anti-CD86-FITC, anti-CD1a-PE, anti-CD83-allo-phycocyanin. To evaluate intracellular cytokine production in humanmonocytes, stimulated cells were surface stained for anti-CD14-FITCand then permeabilized using the Cytofix/Cytoperm kit (BD Bio-sciences). Intracellular cytokines were revealed by staining the cellswith 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 andstained 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 werestained with anti-Ly6C FITC, anti-MHC class II biotin, anti-CD11b Pacificblue, and anti-CD11c-Pe Cy7. To analyze T cell proliferation by CFSE dilu-tion, the cells were stained with anti-CD4 allophycocyanin and the clonotypicanti-OVA TCR Ab KJ1.26 PE (DO11.10) or the combination of anti-TCRV5PEandV6 biotin (OT-II). Biotinylated Abs were revealed using PerCP-labeled streptavidin. All Abs were obtained from BD Biosciences. Fluorescentevents were acquired using an LSR2 and analyzed using FACSDiva software(BD Biosciences).

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 and21) with 2 g of HPV 16/18 L1 VLPs alone, or formulated with aluminumhydroxide (50 g) or with AS04 (50 g of aluminum hydroxide plus 5 g

FIGURE 1. MPL acts as a TLR4

agonist. A, HEK cells transfected with

tlr4, md2, and cd14; with tlr1 and tlr2;

or with tlr2 and tlr6 were stimulated

with MPL, aluminum hydroxide

(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 (5 105 cells/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).

6188 AS04 INDUCES A TRANSIENT LOCALIZED INNATE IMMUNE RESPONSE

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MPL) in 50 l. Two weeks after the second immunization, spleen cellsuspensions were prepared from pooled mice (n 4, 4 pools of 3 spleensamples/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 orHPV-18 Ag. Supernatants were harvested at 48 h later and tested for thepresence of IFN-, IL-2, and IL-5 using a mouse CBA kit.

Statistics

Statistical analyses were performed on logarithmic transformed data. TheShapiro-Wilk test was used to confirm normality. The ANOVA and theTukeys test were applied to identify differences between treatment groups,except in the analysis of TLR responses in PBMCs and NF-B activity forwhich the two-sided Dunnetts test was applied. Statistical significance was

assigned at the value for p

0.05. For the analysis of Ab titers, statisticalsignificance was assigned at a value for p 0.05 and the difference be-tween the groups was also 2-fold.

ResultsMPL 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

FIGURE 2. AS04 stimulated human APCs but not T cells. A, The proportion of TNF--positive (open bars and symbols) and IL-6-positive (shaded bars

and symbols) CD14

monocytes in human PBMCs (n 4 donors). PBMCs in 5 105

cells/well (96-well plate) were stimulated as in Fig. 1B with mediumor 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 2 105 cells/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 2 105 cells/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.


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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 andaluminum 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

activation

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 ofAg and no MPL could be detected in the lymph nodes, suggesting

that the vaccine components remain primarily local to the site of

injection.

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

(23).

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 activitywas 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 atthe 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.

FIGURE 4. AS04 i.m. injection stimulated NF-B activity only at the injection site. NF-B activity was revealed by measuring luminescence using the

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 Dunnetts test relative to the 0 h time

point.B, NF-B activity was measured in surgically exposed iliac,right andleft inguinal lymph nodes. C, Activity measured in spleen homogenates (n 5 donors).

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 Dunnetts testwith respect to the PBS injections. Data represent geometric mean and symbols represent the data points from each of the mice tested.


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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 generallypreceding 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 thelevels 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).7 The

results therefore indicated that the temporal and spatial constraintson 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

7 The online version of this article contains supplemental material.

FIGURE 5. MPL-containing formu-

lations injected into muscle of C57BL/6

mice stimulated transient cytokine pro-

duction at the site of injection. A, IL-6,

TNF-, CCL2,and CCL3 cytokinecon-

centrations measured in homogenized

and pooled (n

6 mice per group) mus-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.


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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 werealso observed both at 24 and 72 h with these injections (p

0.001) (see supplemental Fig. 2). Such MHC class IIhigh cells

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 increasedat 24 h after injection (see supplemental Fig. 2A).7 Similarly, 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).7 The 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 and2-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

FIGURE 6. MPL-containing formulations stimu-

lated increased DC and monocyte numbers in drainingiliac 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 offluo-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.


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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 BMDCstreated 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. CD11cDCs 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). TheAg-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,

FIGURE 7. MPL- and AS04-mediated activation of DCs is associated with T cell activation. A, BALB/c BMDCs were stimulated at 1 106 cells/ml

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 (10 5 T 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 CFSElow CD4 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 (105 DO11.10 T cells/well in a 96-well plate). Proliferation was measured as the percentage of CFSE low T 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 (5 106 cells/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.


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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

agonist (9).

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.

DiscussionAS04 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 localizedactivation of an innate response, which could explain how a

robust adaptive immune response is achieved in AS04-adju-

vanted vaccines.

Crucial role of MPL in AS04 for the stimulation of an innate

immune response

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, 3436). 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, themost 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 tocytokine-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

TLR4 signaling.

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 onMPL 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 L1VLP 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

hydroxide alone?

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 (4850). 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.


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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 (5456). 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

cells (58).

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.

ConclusionIn 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.

AcknowledgmentsWe 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.

DisclosuresArnaud Didierlaurent, Sandra Morel, Laurence Lockman, Sandra Giannini,

Michel Bisteau, Nathalie Vanderheyde, Daniel Larocque, Marcelle Van

Mechelen, and Nathalie Garcon 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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