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Activated Dendritic CellsHuman Vaccine Adjuvant AS01 Depends on Enhancement of Adaptive Immunity by the

Van Mechelen and Sandra MorelMalissen, Bart N. Lambrecht, Nathalie Garçon, Marcelle Fochesato, Najoua Dendouga, Christelle Langlet, BernardBourguignon, Sandrine Wouters, Kaat Fierens, Michel Arnaud M. Didierlaurent, Catherine Collignon, Patricia

http://www.jimmunol.org/content/193/4/1920doi: 10.4049/jimmunol.1400948July 2014;

2014; 193:1920-1930; Prepublished online 14J Immunol 

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8.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/07/13/jimmunol.140094

Referenceshttp://www.jimmunol.org/content/193/4/1920.full#ref-list-1

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The Journal of Immunology

Enhancement of Adaptive Immunity by the Human VaccineAdjuvant AS01 Depends on Activated Dendritic Cells

Arnaud M. Didierlaurent,* Catherine Collignon,* Patricia Bourguignon,*

Sandrine Wouters,* Kaat Fierens,† Michel Fochesato,* Najoua Dendouga,*

Christelle Langlet,* Bernard Malissen,‡ Bart N. Lambrecht,† Nathalie Garcon,*

Marcelle Van Mechelen,* and Sandra Morel*

Adjuvant System AS01 is a liposome-based vaccine adjuvant containing 3-O-desacyl-49-monophosphoryl lipid A and the saponin

QS-21. AS01 has been selected for the clinical development of several candidate vaccines including the RTS,S malaria vaccine and

the subunit glycoprotein E varicella zoster vaccine (both currently in phase III). Given the known immunostimulatory properties

of MPL and QS-21, the objective of this study was to describe the early immune response parameters after immunization with an

AS01-adjuvanted vaccine and to identify relationships with the vaccine-specific adaptive immune response. Cytokine production

and innate immune cell recruitment occurred rapidly and transiently at the muscle injection site and draining lymph node

postinjection, consistent with the rapid drainage of the vaccine components to the draining lymph node. The induction of Ag-

specific Ab and T cell responses was dependent on the Ag being injected at the same time or within 24 h after AS01, suggesting

that the early events occurring postinjection were required for these elevated adaptive responses. In the draining lymph node,

after 24 h, the numbers of activated and Ag-loaded monocytes and MHCIIhigh dendritic cells were higher after the injection of the

AS01-adjuvanted vaccine than after Ag alone. However, only MHCIIhigh dendritic cells appeared efficient at and necessary for

direct Ag presentation to T cells. These data suggest that the ability of AS01 to improve adaptive immune responses, as has been

demonstrated in clinical trials, is linked to a transient stimulation of the innate immune system leading to the generation of high

number of efficient Ag-presenting dendritic cells. The Journal of Immunology, 2014, 193: 1920–1930.

Many Ags contained in subunit vaccines are inherentlyinert proteins or glycoproteins. A common strategy toboost Ag-specific B and T cell adaptive responses

through immunization is to include an immunostimulanting adju-vant in the vaccine (1–3). The ability of adjuvants to trigger earlyinflammatory signals, to increase Ag uptake, and to activate APCsis thought to be crucial for the efficient induction of adaptive mem-ory immune response to vaccines (4, 5).The Adjuvant System AS01 is a liposome-based adjuvant that

contains two immunostimulants MPL and QS-21 (2). MPL is

a nontoxic derivative of the LPS from Salmonella minnesota andis a TLR4 agonist (6). It can stimulate NF-ĸB transcriptionalactivity and subsequent cytokine production (6). MPL directlyactivates APCs such as dendritic cells (DCs) to produce cytokinesand elevated levels of costimulatory molecules (7–9). QS-21 isa natural saponin molecule extracted from the bark of the SouthAmerican tree Quillaja saponaria Molina (10) (reviewed in Refs.2 and 11). The early evaluations of QS-21 as an adjuvant dem-onstrated that it could promote high Ag-specific Ab responses andCD8+ T cell responses in mice (12, 13) and high Ag-specific Abresponses in humans (14). The CD8+ T cell responses induced bysaponin-based vaccines presumably occurred through the promo-tion of Ag cross-presentation in DCs (15, 16). However, specificreceptors and signaling pathways induced by saponin-based adju-vants have yet to be clearly defined.AS01 is included in a number of candidate vaccines against

infections such as those caused by Plasmodium falciparium(malaria) (17, 18), varicella zoster virus reactivation (shingles)(19), HIV (20, 21), and Mycobacterium tuberculosis (22) forwhich both Abs and T cell immunity are thought to be involvedin protection. During the preclinical and clinical evaluations ofthese candidate vaccines, both Ag-specific Ab and CD4+ T cellresponses have been consistently observed, suggesting that AS01promotes these adaptive responses irrespective of the type of Agused (2). In the case of the malaria vaccine candidate, theseadaptive responses have been associated with protection againstmalaria in various clinical trials (23–26) and have supported theselection of AS01 over other adjuvants (2, 23, 27).Given the known immunostimulatory properties of the AS01

constituents liposome-based MPL and QS-21, the objective of thisstudy was to describe, in mouse models, the early innate immuneparameters induced by AS01 after immunization and to identify

*GlaxoSmithKline Vaccines, 1330 Rixensart, Belgium; †Vlaams Instituut voor Bio-technologie Inflammation Research Center, Ghent University, 9052 Ghent, Belgium;and ‡Centre d’Immunologie de Marseille-Luminy, Aix-Marseille Universite, INSERMU1104, Centre National de la Recherche Scientifique Unite Mixte de Recherche7280, 13288 Marseille cedex 9, France

Received for publication April 11, 2014. Accepted for publication June 9, 2014.

This work was supported by GlaxoSmithKline Biologicals SA.

A.M.D., C.C., S.W., K.F., M.F., N.D., C.L., B.M., B.N.L., and S.M. were involved inthe conception and design of the study; C.C., S.W., K.F., and M.F. acquired the data;and A.M.D., C.C., P.B., S.W., K.F., M.F., N.D., B.N.L., N.G, M.V.M., and S.M.analyzed and interpreted the results. All authors were involved in drafting the man-uscript or revising it critically for important intellectual content. All authors had fullaccess to the data and approved the manuscript before it was submitted by thecorresponding author.

Address correspondence and reprint requests to Dr. Arnaud M. Didierlaurent, Glaxo-SmithKline Vaccines, Rue de l’Institut 98, 1330 Rixensart, Belgium. E-mail address:arnaud.x.didierlaurent@gsk.com

The online version of this article contains supplemental material.

Abbreviations used in this article: BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-inda-cene; DC, dendritic cell; dLN, draining lymph node; DT, diphtheria toxin; DTR,diphtheria toxin receptor; gE, glycoprotein E; ILN, iliac lymph node; MHCII,MHC class II.

Copyright� 2014 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/14/$16.00

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relationships between these parameters and the Ag-specific Ab andT cell responses raised against two model Ags, glycoprotein E [gEfrom varicella zoster virus (19)] and OVA.

Materials and MethodsVaccine formulations

The Ags (clinical-grade) gE (19), Alexa Fluor 647–labeled gE, and theAdjuvant System AS01 were produced at GlaxoSmithKline. One humandose of AS01 contains 50 mg MPL (GlaxoSmithKline), 50 mg QS-21(Antigenics, a wholly owned subsidiary of Agenus, Lexington, MA), andliposomes. OVA was obtained from Calbiochem and confirmed to be en-dotoxin free. Alexa Fluor 647–labeled gE and OVA were produced usingthe Alexa Fluor 647 Protein Labeling Kit from Invitrogen, according to themanufacturer’s instructions.

Mouse immunizations

Animal husbandry and experiments were ethically reviewed and carriedout in accordance with European Directive 2010/63/EU and the Glaxo-SmithKline Biologicals Policy on the Care, Welfare and Treatment ofAnimals. C57BL/6 mice were purchased from Harlan Horst, and OTI andOTII mice were purchased from Charles River Laboratories. The i.m.injections were performed in 6- to 8-wk-old femalemice, in both hind limbs,in either the gastrocnemius or tibialis (for histochemistry) muscles and ina volume of 10 ml/muscle (unless stated otherwise).

Spatiotemporal relationship between AS01 and Ags onadaptive responses

Mice (n = 26/group or n = 16/group) were immunized at days 0 and 28with different injection regimens where AS01 (1 mg QS-21 and 1 mgMPL or 5 mg QS-21 and 5 mg MPL in 25 ml), which represents 1/50thand 1/10th of the human doses of AS01, respectively, and gE (1 or 5 mgin 25 ml) were either mixed together (50 ml) or injected separately (25 mleach). Ag alone (1 or 5 mg) was injected in 50 ml. The i.m. injections weregiven at the same time or at separate times (with AS01 being injected from1 h to 7 d before the Ag) at the same site or at contralateral sites into thegastrocnemius muscle.

Ag-specific Ab and T cell response

Anti-gE and anti-OVA Ab concentrations (in ELISA units per milliliter anddefined by internal standards) were measured by ELISA, following a pro-tocol described previously (19), with a minor addition: for the measure-ment of anti-OVA Ab concentrations, 96-well ELISA plates were coatedwith an overnight incubation at 4˚C of 10 mg/ml OVA in PBS.

To assess Ag-specific T cell response, splenocytes from immunized micewere stimulated in vitro using 107 cells/ml (96-well microplate) with gE orOVA peptide pools of 15-mer with 11-aa overlap at 1.25 mg/ml (fromEurogentec) and 1 mg/ml (from Neosystem), respectively. Anti-CD49d andanti-CD28 Abs (1 mg/ml) were added to the culture, and the cells wereincubated 2 h at 37˚C. Brefeldin A (1 mg/ml; BD Biosciences) was thenadded to inhibit cytokine secretion, and cells were further cultured over-night at 37˚C. Cell staining was performed as described previously (19).

For the experiments with the mice with chimeric bone marrow cells (seebelow), flow cytometry was performed with a similar protocol with somemodifications. The following Abs were used for extracellular staining(30 min at 4˚C), anti-CD8 PB (eBioscience), anti–CD3-APC-Cy7 (BDBiosciences), anti–CD4-PE-Tr (Invitrogen), anti-CD44.2 AF700 (104;eBioscience), anti–FcyRII/III-2.4G2 (Bioceros BV), and eF450 dead-cellidentifier (eBioscience). Subsequent intracellular staining was performedusing the Abs, anti–IFN-g-PE-Cy7 (eBioscience), and anti–IL-2-allophyco-cyanin (BD Biosciences) on cells that were washed, fixed with 4% para-formaldehyde for 15 min, and permeabalized with 0.5% saponin (Sigma-Aldrich) for 20 min. The samples were analyzed with the LSRII flowcytometer (BD Biosciences) and FlowJo software with Boolean gates.

Imaging

Fluorescent AS01 was prepared by labeling QS-21 with the fluorophoreBODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). To label QS-21, anester derivative of QS-21 (using the glucuronic acid group) was prepared bytreating QS-21 (in dimethylformamide) with sulfo-N-hydroxy succinimideand dicyclohexylcarbodiimide, as described previously (13). The QS-21-sulfo-NHS ester derivative was purified by precipitation with ethyl acetate,then dried and suspended in 0.1 M NaHPO4 buffer (pH 7), and reacted withan excess of BODIPY FL-EDA (which bears an amine group, D2390; LifeTechnologies). The reaction product, QS-21-BODIPY, was purified by

semipreparative HPLC and isolated as a powder. The biological activity ofQS-21-BODIPY was maintained but lower than the native QS-21 (data notshown).

Frozen cryostat sections of tibialis muscle (10 mm) and draining lymphnode (dLN) (5 mm) from mice immunized with gE-Alexa Fluor 647/AS01-BODIPY and were fixed in 4% paraformaldehyde (muscle) or acetone(dLN). Ab staining of dLNs (to detect T cells, Armenian hamster anti-mouse CD3ε-PE; and B cells, biotinylated rat anti-mouse CD45R/B220;BD Biosciences) was performed in PBS/2% donkey serum, overnight at4˚C, in conjunction with Dylight594-conjugated streptavidin (for CD45R/B220). Tissue sections were examined with a confocal laser scanningmicroscope (Zeiss LSM510/780 meta). Minor lightness and contrast adjust-ments were made using Zeiss and/or other routine image-manipulation soft-ware and were applied uniformly to the whole image.

Innate cell phenotyping in muscle and lymph node

Pooled tissues (gastrocnemius muscle or iliac lymph node [ILN]) fromthree immunized mice were first treated by mechanical dissociation in 3 mlDMEM containing DNase I (100 mg/ml; Roche), 1% FCS and Liberase(Roche) at 0.1 U/ml (muscle), or 0.26 U/ml (ILN) for 30 min under agi-tation at 37˚C (muscle) or at room temperature (ILN). Liberase digestionwas stopped by adding 10 mM EDTA and incubating on ice. Largerclumps of material were removed by passing the preparation througha 100 mM nylon cell strainer (BD Biosciences). The muscle preparationwas enriched for hematopoietic cells by centrifugation on a Percoll gra-dient (Amersham-Pharmacia). Cells were washed twice and resuspendedin PBS containing 2 mM EDTA and 2% FCS.

After treatment with 2.4G2 Ab for 5 min to block the FcR, the cells werestained with the following anti-mouse Abs: anti–Ly6C-FITC, the PE-conjugated lineage identifiers (anti-SiglecF, anti-CD8a, anti-CD4, anti-CD19, and anti-NK1.1), anti–CD86-PE, anti–CD40-PE, anti–CD64-PE, anti–Ly6G-PerCP, anti–Ly6C-PerCP, anti–MHC class II (MHCII)(I-A/I-E)-Alexa700, anti–CD11b-PB, anti–CD11c-PECy7, allophycocyanin-Cy7–conjugated lineage identifiers (anti-CD3, anti-CD19, and anti-Ly6G),and anti–CD45-PO. All Abs were obtained from BD Biosciences. Fluo-rescent events were acquired using an LSR2 and analyzed using FACSDivasoftware (BD Biosciences). Monocytes were defined as Ly6ChighCD11b+

Ly6G2 cells, and neutrophils were defined as SSChighCD11b+Ly6G/Gr1high. After exclusion of neutrophil, monocyte and lymphocyte pop-ulations, DCs were gated as CD11c+MHCII+ from muscles or as CD11cmid

MHCIIhigh from ILNs.

Cytokine detection in muscle and dLNs

For the assessment of proinflammatory cytokine production, the rightgastrocnemius muscle and right iliac lymph node were collected afterimmunization at the indicated time points and stored at280˚C until furtheranalysis. Organs were pooled from three sets of three mice and four sets offour mice immunized with gE+AS01 or gE alone, respectively. Organswere then homogenized and cytokines were measured in supernatants asdescribed previously (8). Briefly, the homogenates were cleared bycentrifugation and stored at 270˚C until analysis. Protein levels weremeasured by cytokine bead array (for TNF-a and IL-1b) or by cytokine-specific beads for all other cytokines using the Luminex platform (Milli-pore). The levels detected for each cytokine are reported in nanograms orpicograms per organ within the homogenate supernatant of the differenttissues.

Evaluation of CD11c-diphtheria toxin receptor transgenicbone marrow mouse chimeras

C57BL/6 mice (8–10 wk old) were sublethally irradiated (8 Gy) and, 4 hlater, received 2 3 106 bone marrow cells i.v. from transgenic (CD11c-diphtheria toxin receptor [DTR]) mouse donors (28).

At least 10 wk after bone marrow reconstitution and 12 h before im-munization, chimeric mice were depleted of CD11c+ cells by an injectionof 200 ng DT or PBS (control) in both gastrocnemius muscles. Chimericmice (n = 6/group) were immunized in both gastrocnemiusmuscles (25 ml/muscle) with OVA (0.5 mg) and gE (2.5 mg) in AS01 or PBS. The im-munization was performed twice 14-d apart.

Ex vivo OTII CD4+ and OTI CD8+ T cell activation

Ex vivo Ag presentation assays were performed using a protocol adaptedfrom Langlet et al. (29). OVA-specific CD8+ T and CD4+ T cells wereisolated from OTI and OTII transgenic mice, respectively, and labeled withCFSE as described previously (19). A total of 25 3 103 OVA-specificT cells were then separately coincubated for 3 d with various numbersof Ly6Chigh monocytes or MHCIIhigh DCs that were purified by cell sorting

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using a FACSAria (.95% purity) from ILNs of mice previously immu-nized (24 h before) with OVA (5 mg)/AS01 in each hind leg. OT-I andOT-II proliferation was determined by the loss of CFSE fluorescence byflow cytometry. As controls, APCs were incubated with MHCI- or MHCII-restricted OVA peptides for 45 min at 37˚C, washed, and incubated with25 3 103 OT-I and OT-II, respectively, at various APC:T ratios as indi-cated, starting with 1:2 ratio.

Statistical analyses

All statistical analyses on cytokine concentrations, Ab concentrations, andT cell frequencies were performed on logarithmic transformed data. TheShapiro–Wilk test was used to evaluate normality. An ANOVA followedby the Tukey’s or Dunnett’s test was applied to identify differences be-tween treatment groups (see figure legends). Statistical significance wasassigned when p # 0.05, and in addition for the analysis of Ab concen-trations, a condition for which the difference between the groups was also.2-fold was applied. Principal component analysis was used to identifycorrelations between cytokine and immune cell responses to immuniza-tion. Absolute quantities and fold differences of repeated measures de-

scribed in Results are derived from geometric means. Principal componentanalysis was performed with SPAD software (version 7; Coheris, Suresnes,France) All other analyses were performed using SAS software (version9.2; SAS Institute).

ResultsLocalization of the vaccine after i.m. injection

Fluorescently labeled QS-21 and Ag were incorporated into the gE/AS01 vaccine to monitor the vaccine’s distribution after i.m. in-jection in situ (Fig. 1). In the muscle, 30 min after injection, QS-21 and the Ag were located in interstitial regions between musclefibers, mainly in the perimysium (Fig. 1A). At 3 and 24 h, QS-21and Ag were detected at much lower levels than at 30 min sug-gesting a rapid clearance from the muscle. Both QS-21 and Agwere also detected in the dLN as early as 30 min, suggestinga rapid drainage from the injection site to the dLN (Fig. 1B). At

FIGURE 1. (A and B) Localization of fluorescently

labeled vaccine components after 30 min, 3 h, and 24 h

after immunization with gE-Alexa Fluor 647/AS01

(5 mg gE-Alexa Fluor 647, and AS01 included 5 mg

MPL and 5 mg QS-21-BODIPY) in 10-mm [muscle

(A)] and 5-mm [dLN (B)] sections. (A) QS-21-BODIPY

(green channel) and gE-Alexa Fluor 647 (red channel).

(B) QS-21-BODIPY (green channel), gE-Alexa Fluor

647 (red channel), B cells (blue channel), and T cells

(white channel). Scale bars, 50 mm (A), 200 mm (B).

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30 min, QS-21 was mainly detected in the subcapsular region ofthe dLN, whereas at 24 h, it was also detected in the medulla. Incontrast to QS-21, the Ag was detected deep within the cortical andmedullary sinuses at 30 min but was barely detectable at 24 h atthe resolution used in this study. Although these data should beinterpreted with caution because QS-21-BODIPY may not fullyreflect the behavior of unlabeled QS-21 and the whole AS01 for-mulation, the differences in the localization and dynamics of QS-21 and Ag suggested that the two components were not physicallyassociated and had different pharmacokinetic properties. Thisconcurred with biochemical analyses, which indicated that AS01was indeed not physically associated with the Ag in the vaccineformulation (data not shown). Because MPL and QS-21 are in-cluded in the same liposome, it is possible that MPL has a similardistribution as QS-21, but this was not assessed in this study.

Rapid and transient induction of cytokines by AS01 at theinjection site and dLN

The kinetics of the innate immune response was examined bymeasuring a panel of representative proinflammatory cytokines andchemokines, as well as the main innate immune cell subset in themuscle and dLN, over a period of 7 d after the injection of gE/AS01or gE.The peak induction of all the cytokines considered together

typically occurred within 24 h after gE/AS01 injection and wassubstantially greater ($8.7-fold) than after Ag injection alone(Fig. 2A). The overall cytokine concentration in the muscle washigher (up to 16-fold at 16 h) than in the dLN. The rapid inductionof cytokines by 3 h at the muscle injection site and dLN suggestedthat cytokines in both locations were induced by resident cellsdirectly stimulated by AS01. This is consistent with a rapiddrainage of AS01 from the injection site to the dLN as suggestedby the confocal imaging (see Fig. 1). The cytokine levels declinedby 48 h and returned to baseline at day 7 (= 168 h), demonstratingthe transient nature of the response (Fig. 2A, 2B). More spe-cifically, the pattern of cytokines production was dominated by che-mokines rather than proinflammatory cytokines, with monocyteand granulocyte-attracting chemokines (e.g., CCL2 and CXCL1,respectively) and T cell–attracting IFN-dependent CXCL9 andCXCL10. Except for IL-6, the other proinflammatory cytokinesexamined, IL-1b (Fig. 2B) and TNF-a, were only induced at lowlevels (,80 pg/organ). Although most cytokines peaked at earlytime points (either 6 or 24 h) and subsided rapidly (e.g., CXCL1and IL-6), others subsided only between days 2 and 7 (e.g.,CXCL9 and CCL2) (Fig. 2A, 2B). Furthermore, neither IL-12p70nor IFN-a was detected above baseline levels (data not shown).The pattern of the inflammatory response was similar overall be-tween muscle and dLN, except for CXCL9 and CXCL10, whichwere more rapidly increased at 3 h in the dLN than in themuscle.These data showed that AS01 induced an inflammatory response

at the injection site and dLN, characterized by the production ofchemokines and IFN-pathway–related cytokines. This inflamma-tory response was transient and subsided by 7 d.

AS01 promotes the transient recruitment of innate cells in themuscle and dLN

Immune cell numbers were evaluated by enzymatic digestion ofmuscle injection sites and dLNs, followed by cell phenotypingusing flow cytometry (Fig. 3).In the muscle and consistent with the chemokine profile (see

Fig. 2), monocyte (Lin2CD11b+Ly6Chigh) and neutrophils (SSChigh

Ly6G+) were the most prominent immune cell types whose num-bers were transiently elevated after the injection of gE/AS01

compared with Ag alone (Fig. 3A, 3B). Lymphocytes numberswere relatively low during the period investigated. At 3 h, thenumber of neutrophils was 9.5-fold higher with gE/AS01 thanwith Ag alone, and at 6 h, the numbers of monocytes and neu-trophils peaked and were 4.9- and 13-fold higher, respectively,with gE/AS01 than with Ag alone. In comparison, the number ofDCs (Lin2, CD11c+MHCII+) was low with no significant changesduring the period investigated. At day 7, the respective numbers ofimmune cells were not different after the injection of gE/AS01 orAg alone, suggesting that the local inflammatory reaction at theinjection site fully resolved by 7 d.In the dLN and at 6–48 h, the total number of immune cells

was greater after the injection of gE/AS01 than after Ag alone

FIGURE 2. AS01 induced a rapid and transient production of cytokines

at the injection site and dLNs. (A and B) Geometric mean cytokine con-

centrations (n = 3); in muscle (injection site; left graphs) or in draining

lymph node (right graphs) homogenates at 3, 6, 24, 48, and 168 h (= day 7)

postimmunization of AS01 (1 mg MPL and 1 mg QS-21)-adjuvanted or

unadjuvanted (No adj.) gE-Alexa Fluor 647 (2 mg) Ag. (B) Both geometric

mean cytokine concentrations (black circles, gE/AS01; gray squares, gE/

[No adj.]) and individual cytokine concentrations (gray circles, gE/AS01;

white squares, gE/[No adj.]) are described for selected cytokines.

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(Fig. 3C, 3D). A 3.4- to 4-fold increase in lymphocytes was observedas early as 3 h after injection. In contrast to the muscle, the numberof myeloid cells only represented a minority of the total population(1–7%) in the dLN. However, the numbers of neutrophils, Ly6Chigh

monocyte, and DCs (defined as CD11cmid MHCIIhigh cells, fur-thermore defined as MHCIIhigh DCs) were higher by up to 53-, 230-,and 8.6-fold, respectively, after the injection of gE/AS01 comparedwith Ag alone (Fig. 3D). The early increase in the number of neu-trophils at 3 h was consistent with the rapid appearance of neutrophilchemoattractants (CXCL1; see Fig. 2). At day 7, lymphocytesaccounted for the remaining increase in cell numbers in the dLN,whereas myeloid cell numbers returned to baseline levels. The in-crease in the number of DCs was apparent from 16 h and consistentwith the migration of muscle resident DCs into the dLN observed byLanglet et al. (29) after the i.m injection of alum.Multicomponent analysis was further performed to correlate

the kinetics of the various cytokines with cell numbers (Fig. 3E,Supplemental Table I). Neutrophil kinetics in the muscle wasassociated (correlation coefficient r . 0.5) with a number ofcytokines, including IL-6, TNF-a, and IL-1b, as well as twoknown neutrophil-attracting cytokines (CXCL2 and CXCL1),suggesting that early proinflammatory signals were associatedwith rapid neutrophil migration to the injection site. However, inthe dLN, neutrophil kinetics was associated with a different butoverlapping set of cytokines than in the muscle, suggesting a dif-ferent mechanism regulating the increase in neutrophil number inthe dLN. In contrast to neutrophil kinetics, the kinetics of the otherimmune cell types in the muscle and dLN were not so clearlyassociated with any of the cytokines examined, suggesting that thebehavior of these cells was governed by a more complex pattern ofsignals. In particular, monocyte recruitment did not seem to beassociated with CCL2 in this model.

Spatiotemporal colocalization of AS01 and the Ag is necessaryfor adjuvant effect

To investigate the time window in which the kinetics of the innateimmune responses relate to the adaptive immune responses, AS01was first injected followed by gE at different intervals [from 0 to72 h at the same location in the muscle (Fig. 4, left graphs)]. Twoweeks after immunization, Ag-specific Ab and T cell responseswere measured. When the Ag and AS01 were injected at the sametime in the same site, either separately or premixed, the respectiveAb and T cell responses were not different. However, these Ab andT cell responses were .13,000- and .5.5-fold higher, respec-tively, than after the injection of Ag alone. The adaptive immuneresponses were also high when the Ag was injected 1 h after AS01instead of at the same time. However, the adaptive responses weresubstantially lower when the Ag was injected 24 or 72 h afterAS01 (Ab responses, 6- and 540-fold lower, respectively, and Tcell responses, 5.3- and 6-fold lower, respectively). With the 72-hseparation between injections, the T cell response was not dif-ferent from that induced by Ag alone, although the Ab responsewas 24-fold higher than that induced by Ag alone. However, thisdifference in Ab responses was not observed when 5-fold loweramounts of AS01 and Ag were used (Supplemental Fig. 1).To investigate the spatial relationship between the innate im-

mune responses and the adaptive responses, the Ag was injectedin the limb contralateral to that in which AS01 was injected(Fig. 4, right graphs). Ag-specific Ab and T cell responses were

FIGURE 3. AS01 induced a transitory increase in innate immune cell

numbers in the muscle (A and B) and dLN (C and D), 3–168 h (= day 7)

postimmunization of AS01 (1 mg MPL and 1 mg QS-21)-adjuvanted or

unadjuvanted (No adj.) gE-Alexa Fluor 647 (2 mg) Ag. (A–C) Geometric

mean cell number/tissue (n = 3) of (CD45+) immune cells including

monocytes (Lin2CD11b+Ly6Chigh), neutrophils (SSChighLy6G+), and DCs

(CD11c+MHCII+ in muscle and CD11c+MHCIIhigh in the dLN). (E) The

immune cell and cytokine kinetics (shown in Fig. 3) were examined using

principal component analysis (PCA). The principal components 1, 2, and 3

(represented by the x, y, and z axes, respectively, in the three-dimensional

scatter plot) accounted for 73 and 88% of the variation in the injection site

(muscle; upper scatter plot) and dLN (lower scatter plot) data sets, re-

spectively. Neutrophils (N) are depicted by cyan triangles. Other cell types

are depicted by gray triangles (B, B cells; mDC, MHCIIhigh DCs; rDC,

MHCIImed DCs; M, monocytes; and T&NK, T and NK cells). Cytokines

clusters with potential associations with neutrophils are depicted by purple

circles (see also Supplemental Table I) and include (IL-6, IL-1b, TNF-a,

CXCL1, CXCL2, CXCL10, CCL2, and CCL3 in muscle and IL-1b, TNF-a,

CXCL2, CXCL9, CXCL10, CCL2, CCL3, CSF1, and CSF3 in the dLN).

Other cytokine clusters are depicted by green circles.

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substantially lower (110- and 4.4-fold, respectively) when gE wasinjected in the contralateral site rather than the same site as AS01(Fig. 4, right graphs). Although the T cell response was not dif-ferent from that when Ag was injected alone, the Ab response wasslightly higher. However, this difference in Ab responses was notobserved when 5-fold lower amounts of AS01 and Ag were used(Supplemental Fig. 1).These results indicated that maximal Ag-specific T cell and Ab

responses required the Ag to be injected at the same site togetherwith AS01 and within 24 h. This is consistent with the rapidclearance of Ag and adjuvant at the injection site, as shown byconfocal imaging (Fig. 1). This also suggested that elements of theinnate immune response that were triggered by AS01 within 24 hand local to the injection site potentially contributed to the in-creased magnitude and quality of the adaptive responses. Thislimited time window seemed to be more critical for the inductionof T cell response than for Ab response.

AS01 enhances the recruitment and activation of APCs in thedLN

The increased activity of APCs in the dLN could explain how theactivation of innate immunity by AS01 translates into enhancedAg-specific adaptive immune responses. Ag-carrying innate im-mune cells were identified using fluorescently labeled gE Ag, eventhough the method could have underestimated their number be-cause of the anticipated loss of fluorescence with Ag degradationafter uptake (Fig. 5A, 5B). Higher numbers of Ag+ monocyte,

MHCIIhigh DCs, and neutrophils were detected up to 48 h afterimmunization with gE/AS01 than with Ag alone (Fig. 5A). Atearly time points, neutrophils were the main Ag-loaded cells, buttheir numbers rapidly subsided. After 24 h, Ag+ monocytes were10 times more frequent than Ag+MHCIIhigh DCs. Also Ag+

monocytes were 22% of all monocytes, whereas Ag+MHCIIhigh

DCs were 4% of all MHCIIhigh DCs. In contrast to other adjuvantssuch as alum or emulsion in the same model (8, 30), AS01 did notincrease the amount of Ag detected per cell as measured by meanfluorescence intensity of Ag+ cells (Fig. 5B). However, monocyteand MHCIIhigh DCs expressed higher levels of the costimulatorymolecule CD86 and CD40 at 24 h after the injection of gE/AS01than after Ag alone (Fig. 5C). The expression of CD40 and CD86returned to baseline levels at day 7, consistent with the transientnature of the AS01-dependent innate immune response. Therefore,coadministration of AS01 with the Ag increased the number ofAg-carrying and activated APCs without a clear increase in theircapacity to take up Ag.Both DCs and monocyte populations are heterogeneous

(Fig. 5D) (31, 32). At 24 h after immunization with gE/AS01,a substantial proportion of Ly6Chigh monocytes expressed CD11cand MHCII, showing that AS01 affected the differentiation ofmonocytes in situ. The MHCIIhigh DCs population contained cellsthat expressed CD11b and CD64 that are both markers ofmonocyte-derived DCs (33), suggesting that, in addition to theclassical CD24+ DCs (also known as CD8a+ DCs) and CD11b+

CD642 DCs, a proportion of DCs were derived from monocytes(29, 34). Given that those Ly6C2CD64low monocyte-derived DCswere part of the MHCIIhigh population (see Fig. 5D), theyexpressed higher levels of MHCII than Ly6Chigh monocytes. Thistransition of monocytes into DCs was more apparent in the dLNsfrom mice injected with gE/AS01 than with gE alone (i.e., 13%compared with 4% of CD11b+CD64+ cells in the Ly6C2CD11c+

MHCIIhigh population). Whether these differentiated monocytesoriginated from the muscle after extravasation or directly fromblood was not assessed.In the dLN after the injection of gE alone, the CD11c+ DCs could

also be resolved into two distinct populations by the relative ex-pression level (medium or high) of MHCII. The MHCIImedium

population (Fig. 5D, asterisk in nonadjuvant group) reflected aLN-resident DC population that was not apparent 24 h after theinjection of gE/AS01, suggesting that AS01 may have directlyinduced higher levels of MHCII in this population. Therefore,AS01 broadened the heterogeneity of MHCIIhigh DCs populationin the dLNs to contain bona fide DCs (either resident or migratingfrom the muscle injection site) and monocyte-derived DCs.

Improved T cell response by AS01 is mediated by MHCIIhigh

DCs

To examine the ability of the different APC subsets to present Ag tocognate T cells, two murine models were used (5): one thatmeasured responses after DT-mediated CD11c+ cell depletion anda second that measured the ability of purified APC population topresent OVA to cognate–T cell ex vivo. OVA was confirmed asa suitable model Ag because i.m. immunization with OVA/AS01compared with OVA alone increased OVA-specific CD4+ andCD8+ T cell frequencies (Supplemental Fig. 2A). Restimulationwith OVA peptide pools of splenocytes from OVA/AS01-immunizedmice also indicated that both Th1 (IFN-g) and Th2 cytokines (IL-5and IL-13) were induced, although the cytokine response waslargely shifted toward Th1 compared with OVA/Alum-immunizedmice (Supplemental Fig. 2B).CD11c+ cell depletion was achieved by injecting DT into ani-

mals that had bone marrow transplants from mice transgenic for

FIGURE 4. High Ab and T cell responses were dependent on the Ag

being injected at the same site and within 24 h after AS01. Ag-specific Ab

concentrations (upper graphs; n = 26, left; n = 16, right) and CD4+ T cell

responses (percentage of gE-specific CD4+ T cells with respect to all CD4+

T cells; lower graphs; n = 13, left; n = 8, right) in splenocyte cultures

derived from mice subject to different injection regimes. The cytokine

expression profile (IL-2, IFN-g, or both) of the gE-specific CD4+ T cells

are indicated by differences in shading. The values from mice injected with

AS01 and Ag together in the same syringe are indicated by hatched bars.

The adaptive responses were measured 30 d after the second immuniza-

tion. The Ag was 5 mg gE and AS01 included 5 mg MPL and 5 mg QS-21.

Histograms represent geometric means, and error bars represent 95%

confidence intervals. Highlighted statistical relationships are indicated by

horizontal lines, either between the coinjection of AS01 and Ag and other

regimes (black lines) or between the injection of Ag alone and other

regimes (gray lines). ***p , 0.001.

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DTR driven by the CD11c promoter (35). Transgene expressionrendered the cells sensitive to the lethal effect of DT.Using an immunization protocol combining both Ags, gE and

OVA, gE-specific and OVA-specific CD4+ and CD8+ T cellresponses were higher with gE-OVA/AS01 than with gE-OVAalone (Fig. 6A), in line with the other immunizations in wildtype (WT) mice (see Fig. 4, Supplemental Fig. 2A, 2B). The in-jection of DT 12 h before gE-OVA/AS01 immunization abrogatedboth CD4+ and CD8+ OVA and gE-specific responses (Fig. 6A),

indicating that CD11c+ cells were necessary for the induction ofthe Ag-specific T cell responses to vaccines adjuvanted with AS01.Because both MHCIIhigh DCs and a subset of Ly6C+ monocytes

express CD11c (see Fig. 5D), the ability of those two APC subsetsto directly present Ag (OVA) to T cells was assessed ex vivo usingOTII and OTI T cells. Cells were isolated by cell sorting fromdLNs 24 h after immunization with OVA/AS01 (SupplementalFig. 2C, 2D). As shown for the gE Ag in Fig. 5A, the propor-tion of OVA+ cells after injection of fluorescent OVA/AS01 had

FIGURE 5. DCs and monocytes were activated and

took up Ag after immunization. (A) Geometric mean

Ag+ cell number/dLN (n = 3) of monocyte (CD11b+

Ly6Chigh), DCs (CD11c+MHCIIhigh), and neutrophils

(SSChighLy6G+), 3–168 h after immunization with gE

(2 mg; No adj.) or gE/AS01 (AS01; 1 mg MPL and

1 mg QS-21). (B) Geometric mean Ag staining inten-

sity (mean fluorescence intensity [MFI], n = 3) of

monocytes and DCs, 24 h after immunization with gE

(No adj.), or gE/AS01 (AS01). (C) Geometric mean

CD86 staining intensity (MFI, n = 3) of monocytes and

DCs 24, 48, and 168 h after immunization with gE (No

adj.) or gE/AS01 (AS01). (D) Representative flow

cytometry outputs of monocyte and DCs taken from the

dLN 24 h postimmunization with gE (No adj.) or gE/

AS01 (AS01) revealing heterogeneous populations of

cells, based on further subdivisions according to the

expression of CD11c and MHCII (within monocytes

Ly6Chigh) and migratory DCs (CD11b, CD24, and

CD64). The geometric mean percentage (and SD) of

MHCIIhigh-DC numbers within a given phenotyped

subpopulation over all MHCIIhigh DCs is indicated

adjacent to the respective outlined subpopulation and

was calculated from four repeat cell-sorting runs

using the same source material. *Note that a pop-

ulation of resident DCs was only clearly identified

with the CD11c+MHCIImed gate after immunization

with gE without AS01.

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been found to be higher in the monocyte population than in theDC population (25–34 versus 4–9%). OTII- or OTI-proliferationafter 3 d of coculture (measured by the decrease of the CFSEfluorescent cytosolic dye) was used as a readout of Ag presenta-tion. MHCIIhigh DCs from mice immunized with OVA/AS01substantially induced both CD4+ and CD8+ T cell proliferationin a T cell:DC ratio dependent manner (Fig. 6B, 6C). In contrast,Ly6C+ monocytes (which excludes monocyte-derived DCs) frommice immunized with OVA/AS01 did not induce significant CD4+

T or CD8+ T cell proliferation at any ratio. Pulsing those mono-cytes with OVA peptides restored CD8+ but not CD4+ T cellproliferation (data not shown), demonstrating that these cellswere not able to present Ag to CD4+ T cells, even in the presenceof excess peptides loaded onto MHCII molecules.This suggests that AS01 enhanced the ability of MHCIIhigh DCs

to present Ag to T cells but not monocytes, even though mono-cytes were the main cell type carrying Ag in the dLN (seeFig. 5A), and expressed MHCII and costimulatory molecules (seeFig. 5C, 5D, Supplemental Fig. 2C).Therefore, because AS01 increased the number of MHCIIhigh

DCs in the dLN, as well as their ability to efficiently prime T cells,these DCs were likely to be responsible for most of the T cellresponse after immunization with an AS01-adjuvanted vaccine.T cell activation did not appear to require an increase in Agloading per se but was associated with the increased activation ofDCs, as suggested by the elevated expression of CD40 and CD86.Although Ly6C+ monocytes were unable to efficiently activateT cells, DCs derived from monocytes (Ly6C2CD11c+MHCIIhigh

CD11b+CD64+) that are included in the MHCIIhigh DC populationmay have contributed to T cell priming.

DiscussionAdjuvant System AS01 contains two immunostimulants, MPL andQS-21, in a liposome formulation. After its injection, the expectedimpact on the innate immune response was clearly demonstrated inthe mouse models used in this study. This innate immune responsewas rapid and transient in both the injected muscle and dLN, andreflected to a large degree, the rapid drainage of the Ag and theadjuvant from the muscle to the dLN. The significance of this innateresponse was supported by the observation that the enhanced Ag-specific B and T cell responses were largely dependent on the Agbeing injected at the same location as AS01 within 24 h. Severalinnate immune parameters induced by AS01 may explain its abilityto promote adaptive immune responses.First, AS01 increased the number of APCs that were efficient at

priming T cells in the dLN. These APCs were within the MHCIIhigh

DC population and were identified in ex vivo presentation assaysand in an in vivo transgenic CD11c+ cell depletion model. Amongother mechanisms, the activation of this DC population, reflectedby elevated expression levels of costimulatory molecules, mayhave directly contributed to T cell–priming efficiency (36, 37).However, unlike other adjuvants such as oil-in-water emulsions,MPL, or alum-containing adjuvant AS04 (8, 30, 38), AS01’s ad-juvant effect was not associated with an increased uptake of Ag by

FIGURE 6. T-cell responses to immunization were dependent on

CD11c+ MHCIIhigh DCs. (A) Geometric mean Ag-specific T cell responses

(n = 6/group) 7 d after the second immunization with gE + OVA or gE +

OVA/AS01 (OVA, 1 mg; gE, 5 mg; and AS01 included 5 mg MPL and 5 mg

QS-21) in wild type mice containing bone marrow transplants from

transgenic (CD11c-DTR) mice and treated with (+) or without (2) in-

jection of DT 24 h before immunization. (B) Representative flow cytom-

etry histograms of OTII CD4+ T cell and OTI CD8+ T cell activation

measured in terms of loss of cell cytosolic CFSE fluorescence (because of

proliferation) after ex vivo coculturing with DCs (CD11c+MHCIIhigh) or

monocytes (CD11b+Ly6chigh) taken 24 h after immunization with OVA/

AS01 (5 mg OVA and AS01 included 1 mg MPL and 1 mg QS-21) in

different ratios starting at two T cells to one APC. Green and purple areas

under the curves denote proliferating and nonproliferating cell populations,

respectively. (C) Quantification of OT-II and OT-I cell divisions with re-

spect to different ratios of T cells to APCs.

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DCs or with an Ag depot effect. The DC population includedtissue-resident DCs that migrated to the dLN upon activation andmonocyte-derived DCs, similar to what has been shown beforewith a LPS adjuvant, as well as with alum adjuvant injected viathe peritoneal route (5, 29, 39). Indeed, MPL in AS01 may havedirectly activated muscle-resident DCs (8) and could also haveenhanced the differentiation and migration of monocyte-derivedDCs (29).Second, the rapid and concomitant free-flow of AS01 and Ag to

the dLN was likely to have affected the magnitude of the adaptiveresponse. Resident DCs in the dLN may have been directly acti-vated by AS01 (or indirectly by the inflammatory milieu) and maytherefore provide an early signal to cognate T cells, before theaccumulation of the migratory DCs. The rapid activation of DCs ata stage when the Ag was most readily available to be endocytosed(i.e., soon after immunization) may be an additional key factor inestablishing a strong T cell response. This is suggested by Kamathet al. (40) who recently demonstrated that, in the converse situa-tion, the early recruitment of nonactivated Ag-loaded APCs within the dLN may be detrimental for Th1 responses. Given that highadaptive responses were achieved even when Ag was injectedhours after AS01, our study shows that high adaptive responsescan be associated with a rapid induction of inflammation and APCactivation and may not require a physical association betweenadjuvant and Ag.Third, in the dLN, the rapid recruitment of naive T cells was

likely to have further enhanced the frequency of T and APCinteractions (41), thereby supporting Ag presentation. This may beexplained by the early activation of resident DCs in the dLN, asshown by others (42). The CXCR3 ligands CXCL9 and CXCL10are often associated with Th1 responses (43) and were prominentamong the cytokines detected in the dLN. The expression of bothchemokines by LN stromal cells and DCs may have enhanced Th1responses by creating a favorable environment facilitating T cellpriming at the periphery of the dLN (44).Fourth, other innate immune effectors also may have modulated

the adaptive response. Neutrophils were rapidly recruited at theinjection site and in the dLN after immunization with AS01-adjuvanted vaccine. Although not assessed in this study, neu-trophils have been reported to have either no effect or a detrimentaleffect on the adaptive response (45, 46). Nevertheless, neutrophilsmay play a role in controlling the degree of local inflammationand participate in Ag clearance (47). Monocytes were the largestpopulation of innate immune cells recruited after immunizationwith gE/AS01. Monocytes may play a critical role in the resolu-tion of inflammation at the injection site by differentiating locallyinto macrophages (48). This is supported by the absence ofinflammatory cells and cytokines in the muscle 1 wk post-immunization with gE/AS01, suggesting a full recovery of theinjected muscle. Although some of the Ly6chigh monocytesexpressed MHCII and costimulatory molecules and were loadedwith Ag, most of these cells were unable to prime T cells effi-ciently, in line with previous reports (29, 49, 50). An immuno-modulatory role of those monocytes in AS01 mode of actionremains to be investigated. Indeed, monocytes may have sup-portive roles, such as helping DCs in directing Th1 differentiationof CD4+ T cells (51) or in activating CD8+ memory T cells andNK cells (52), but may have had also a suppressive role, as re-ported recently (53).Both MPL and QS-21 in AS01 have been associated with en-

hancing Ab and T cell responses after immunization (19) and mayaffect the innate immune responses differently. In our study, theinduction of a number of cytokines (e.g., IL-6, CCL2, and CCL3)may have been dependent on MPL (8), whereas the induction of

IL-1b may have been due to QS-21 because IL-1b induction isassociated with the activation of the inflammasome, and the ac-tivation of the inflammasome has been observed with QuilA (54).In addition, QS-21 is likely to have played a role in CD8+ T cellinduction in the mouse model used in this study, perhaps mediatedby a particular population of DCs, as reported recently (35). Thecontributory roles of MPL and QS-21 in the response are currentlyunder investigation.AS01 has been selected in a number of human vaccine candi-

dates because of its association with enhanced immune responses,both humoral and cellular (2, 19, 55). Recently, the RTS,S vaccinewas shown to confer protection of 30–47% against uncomplicatedand severe malaria in young African children and infants, re-spectively (17, 18). Although one difference between mice andhuman is the lack of detectable CD8+ T cell responses in humans(21, 22, 25), the results described here now provide insights intothe role of AS01 in generating protective vaccine responses inhumans, particularly through its ability to generate a strong cel-lular response. The features of AS01’s adjuvant activity that arelikely to be relevant in humans are the transient activation of aninnate response at the site of injection, the induction of a specificpattern of cytokines, and the generation of a heterogeneous DCpopulation in the dLN that is efficient at priming T cells. Hence,the details on AS01’s adjuvant activity identified in this study mayhelp in the further development of human vaccine adjuvants re-quired for promoting Ag-specific cell-mediated responses as wellas Ab responses.

AcknowledgmentsWe thank Nabila Amanchar, Stephanie Quique, Hajar Larbi, Aurelie Chalon,

and Karine Gillard for providing technical support, Kendal Ryter for

generating fluorescent (BODIPY-labeled) QS-21, and Frederic Renaud and

Gael de Lannoy (all at GlaxoSmithKline Vaccines) for performing the

statistical analyses. We thank Sandra Giannini for providing advice with

the design and interpretation of experiments evaluating spatiotemporal

relationships between AS01 and Ag and Catherine Gerard, Caroline Herve,

Margherita Coccia, Lode Schuerman, and Robbert Van der Most (all at

GlaxoSmithKline Vaccines) for providing constructive and scientifically

critical reviews of the manuscript. We also thank Mirjam Kool, Hamida

Hammad, and Martin Guilliams (Vlaams Instituut voor Biotechnologie,

Ghent University) for providing advice in the design and interpretation

of the transgenic (CD11c-DTR) bone marrow mouse chimera experiments,

Matthew Morgan (MG Science Communications) for providing scientific

writing services, and Ulrike Krause (GlaxoSmithKline Vaccines) for pro-

viding editorial advice and coordinating the manuscript’s development.

DisclosuresA.M.D., C.C., P.B., S.W., M.F., N.D., C.L., N.G., M.V.M., and S.M.

are employees of the GlaxoSmithKline group of companies. A.M.D.,

P.B., N.D., N.G., M.V.M., and S.M. own GlaxoSmithKline stocks. N.G.

is listed as an inventor on patents owned by GlaxoSmithKline. B.M., K.F.,

and B.N.L. report no financial conflicts of interest.

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