of September 10, 2018.This information is current as

-GlucanβCells via Dectin-1 by Incorporating Conjugate Vaccine Targeted to DendriticTricomponent Mannan Tetanus Toxoid Enhanced Immunogenicity of a

Ostergaard, David R. Bundle and Nicolas TouretTomasz Lipinski, Amira Fitieh, Joëlle St. Pierre, Hanne L.

http://www.jimmunol.org/content/190/8/4116doi: 10.4049/jimmunol.1202937March 2013;

2013; 190:4116-4128; Prepublished online 20J Immunol 

Referenceshttp://www.jimmunol.org/content/190/8/4116.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2013 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

Enhanced Immunogenicity of a Tricomponent MannanTetanus Toxoid Conjugate Vaccine Targeted to DendriticCells via Dectin-1 by Incorporating b-Glucan

Tomasz Lipinski,*,1,2 Amira Fitieh,†,‡,2 Joelle St. Pierre,x Hanne L. Ostergaard,x

David R. Bundle,* and Nicolas Touret†

In a previous attempt to generate a protective vaccine against Candida albicans, a b-mannan tetanus toxoid conjugate showed poor

immunogenicity in mice. To improve the specific activation toward the fungal pathogen, we aimed to target Dectin-1, a pattern-

recognition receptor expressed on monocytes, macrophages, and dendritic cells. Laminarin, a b-glucan ligand of Dectin-1, was

incorporated into the original b-mannan tetanus toxoid conjugate providing a tricomponent conjugate vaccine. A macrophage cell

line expressing Dectin-1 was employed to show binding and activation of Dectin-1 signal transduction pathway by the b-glucan–

containing vaccine. Ligand binding to Dectin-1 resulted in the following: 1) activation of Src family kinases and Syk revealed by

their recruitment and phosphorylation in the vicinity of bound conjugate and 2) translocation of NF-kB to the nucleus. Treatment

of immature bone marrow–derived dendritic cells (BMDCs) with tricomponent or control vaccine confirmed that the b-glucan–

containing vaccine exerted its enhanced activity by virtue of dendritic cell targeting and uptake. Immature primary cells

stimulated by the tricomponent vaccine, but not the b-mannan tetanus toxoid vaccine, showed activation of BMDCs. Moreover,

treated BMDCs secreted increased levels of several cytokines, including TGF-b and IL-6, which are known activators of Th17

cells. Immunization of mice with the novel type of vaccine resulted in improved immune response manifested by high titers of Ab

recognizing C. albicans b-mannan Ag. Vaccine containing laminarin also affected distribution of IgG subclasses, showing that

vaccine targeting to Dectin-1 receptor can benefit from augmentation and immunomodulation of the immune response. The

Journal of Immunology, 2013, 190: 4116–4128.

Candidiasis is one of the leading causes of hospital-acquiredbloodstream infections. Despite antifungal therapy, atleast 40% of affected individuals die of this disease (1, 2).

The poor outcome of almost half the patients treated with ap-propriate antifungal therapy has spurred the development of pre-ventative measures such as active immunization strategies (3–5).Although a few existing candidate vaccines show promise (6–9),others require aggressive immunization protocols and remain lesssatisfactory (10). We envisaged a conjugate vaccine based on the

protective b-mannan epitope of Candida albicans conjugated to

tetanus toxoid (11), a licensed carrier protein, with a third com-

ponent, a b-glucan, that engages Dectin-1, a C-type lectin receptor

of the dendritic cell (DC) and targets the vaccine to these pro-

fessional APCs.Invasion of opportunistic fungal pathogen, especially in the case

of Candida—a human commensal organism—usually results from

skin and mucosal interface exposure or following clearance of

bacterial flora by antibiotic treatment. Susceptibility to fungal

infection is exacerbated in patients who are immunocompromised,

such as those who had elective abdominal or other surgical pro-

cedures (2) or chronically immunosuppressed in the case of HIV-

positive patients (12). A major challenge in developing an effi-

cacious anti-Candida vaccine is whether vaccination would be

effective in immunosuppressed patients. Given that most of the

therapies used to prevent xenograft rejection lower the acquired

branch of the immune system, we hypothesized that developing

a vaccine that targets the innate immune system would be useful in

fighting Candida infections.The first line of defense against fungal pathogens involves in-

nate immune cells equipped with an arsenal of pattern-recognition

receptors that recognize unique pathogen-associated molecular

patterns. DCs, neutrophils, and macrophages are the main players

in fungal recognition through several receptors that bind yeast cell

wall components. Following binding and internalization of the

pathogen, innate immune cells produce and secrete cytokines, such

as IL-6 and TGF-b, which promote differentiation of naive T cells

into Th17 (13–15). The latter subclass of Th cells is involved in

evoking a cell-mediated response against the invasive fungi, which

is key to defending against the infection (16, 17).

*Department of Chemistry, Alberta Glycomics Centre, University of Alberta,Edmonton, Alberta T6G 2G2, Canada; †Department of Biochemistry, Universityof Alberta, Edmonton, Alberta T6G 2E1, Canada; ‡Biophysics Department, Facultyof Science, Cairo University, 12613 Giza, Egypt; and xDepartment of Medical Mi-crobiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2E1,Canada

1Current address: Institute of Immunology and Experimental Therapy, Polish Acad-emy of Sciences, Wroclaw, Poland.

2T.L. and A.F. contributed equally to this study.

Received for publication October 22, 2012. Accepted for publication February 19,2013.

This work was supported by the Canadian Institutes of Health Research, the Cana-dian Foundation for Innovation, and Alberta Innovates Health Solutions (to N.T.), andby a Natural Sciences and Engineering Research Council Discovery Research grantand the Alberta Glycomics Centre supported by Alberta Innovates (to D.R.B.).

Address correspondence and reprint requests to Dr. Nicolas Touret, University ofAlberta, Department of Biochemistry, Katz Group Rexall Centre, Room 4020H,Edmonton, AB T6G 2E1, Canada. E-mail address: touret@ualberta.ca

Abbreviations used in this article: BMC, bone marrow cell; BMDC, bone marrow–derived dendritic cell; DC, dendritic cell; PBST, PBS containing 0.1% Tween; PFA,paraformaldehyde; SFK, Src family kinase; Treg, regulatory T cell; TS-TT, trisaccha-ride–tetanus toxoid; TS-TT-Lam, TS–TT–laminarin; WT, wild-type.

Copyright� 2013 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202937

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Dectin-1 is a pattern-recognition receptor that has been impli-cated in defending against invasions by C. albicans (18–20), As-pergillus (21), Coccidioides (22), and Pneumocystis (23). Thisreceptor is a single transmembrane protein with a carbohydraterecognition domain in its extracellular region that binds b-1,3-glucan (a major component of the cell wall of a variety of fungalpathogens), a transmembrane segment, and an N terminus cyto-solic domain. Upon binding to b-glucans, the Src family-dependenttyrosine phosphorylation of the ITAM-like motif (abbreviatedhemITAM) in the cytoplasmic tail recruits and activates the adaptorprotein Syk (24). Syk, in turn, triggers downstream effectors, in-cluding NF-kB, which translocates to the nucleus to turn on mRNAtranslation of cytokines. In particular, synthesis and secretion ofIL-6 and TGF-b are induced in a Dectin-1–dependent manner inDCs and macrophages and stimulate Th17 cells.Two C. albicans candidate vaccines based on carbohydrate Ags

have received attention. One vaccine, which is based on b-mannanconjugates, showed potential in eliciting active and passive pro-tection in mice (25–27). Various forms of b-glucan conjugated tothe toxoid CRM-197 induced protection against Candida andAspergilla species (28). The cellular mechanism responsible forthe protective effect of this vaccine conjugate was not investi-gated, but most likely involved an Ab response characterized bythe production of murine (6) and human (29) anti–b-glucan Igmainly of the subclass IgG1 and IgG2.A conjugate vaccine that is composed of synthetic b-mannan

trisaccharide linked to tetanus toxoid (11, 30, 31) achieved sig-nificant reductions in fungal counts in an immunocompromisedrabbit model of disseminated candidiasis (32). This vaccine in-duced high titers of b-mannan–specific Abs, in the range of20,000–80,000, after only two injections using alum as adjuvant(32). Disappointingly, the same vaccine given to mice producedcorresponding titers of 10,000 or lower, and these Ab levels werenot significantly improved by more aggressive immunizationprotocols that employed powerful adjuvants such as CFA (33).However, when an Ag-pulsed DC-based vaccine strategy wasused, b-mannan trisaccharide-peptide conjugates were able toprotect mice in a disseminated candidiasis model of disease (10).This result prompted us to consider targeting the trisaccharide–tetanus toxoid (TS-TT) conjugate vaccine to DCs. We chose tocreate a conjugate vaccine bearing an additional carbohydrate epi-tope, the b-glucan laminarin, which serves as a ligand for Dectin-1receptor (34).We show in this work that incorporating laminarin to the vaccine

results in binding and activation of DCs and elicited significantlyhigher titers against the b-mannan epitope. Given that small oli-gosaccharide epitopes, even when conjugated to powerful carrierproteins, are poorly immunogenic in mice, an ability to enhanceoligosaccharide-specific responses by targeting vaccines to DCsvia carbohydrate-binding receptors has considerable practicalvalue.

Materials and MethodsReagents and Abs

Rabbit anti-phospho Syk (Tyr525/526) and rabbit anti-phospho Syk (Tyr352)were purchased from Cell Signaling Technology. Alexa Fluor 488 conju-gated rabbit anti-phospho Src (Tyr418) was from Life Technologies (GrandIsland, NY). Monoclonal anti-p65 NF-kB was obtained from Santa CruzBiotechnology (Santa Cruz, CA), and mouse anti-actin was from Sigma-Aldrich (St. Louis, MO). Goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, and fluorescently conjugated secondary Abs were purchased fromJackson ImmunoResearch Laboratories (West Grove, PA). Fluorescentlylabeled Abs used for flow cytometry were from eBioscience (San Diego,CA) and included those specific for CD11c, CD11b, CD80, CD86, and F4/80. Anti-human Dectin-1 was from R&D Systems (Minneapolis, MN), and

anti-mouse Dectin-1 was from AbD Serotec. Ultrapure LPS (LPS fromEscherichia coli 0111:B4, g-irradiated) was purchased from Sigma-Aldrich. Mouse rIL-4 was from eBioScience. Laminarin (soluble b-glu-can from Laminaria digitata) and curdlan (from Alcaligenes faecalis) wereboth from Sigma-Aldrich.

b-glucans and vaccine conjugates

Propargylated laminarin preparation. Laminarin (100 mg, soluble b-glu-can from L. digitata; Sigma-Aldrich) was dissolved in 0.2 M phosphatebuffer (pH 6.0; 1 ml) in a 6-ml Kimball glass vial with magnetic stirringbar. Propargylamine hydrochloride (120 mg) was added, followed by so-dium cyanoborohydride (5 mg), and stirred in incubator at 37˚C for 7 d.Additional portions of sodium cyanoborohydride (5 mg each) were addedon days 2 and 4. The reaction mixture was then diluted with water to 10 mland precipitated with 4 vol of ethanol. After centrifugation (7000 3 g, 20min), the precipitate was dissolved in a minimal amount of water andpassed through a Sephadex G-25 column (2.5 3 25 cm). The column waseluted with water, and fractions containing sugar were collected and ly-ophilized. Analysis led to the conclusion that 15% of laminarin moleculeswere substituted with a propargyl group. According to previously pub-lished results, only ∼20% of laminarin molecules in commercial prepa-rations are available for derivatization via the reducing end because themajority of chains are capped by mannitol (35, 36). In this study, we foundthat 15% of laminarin molecules were substituted with a propargyl group(data not shown).

Tetanus toxoid conjugate vaccines (Fig. 1). Tetanus toxoid (36 mg; StateSerum Institute, Copenhagen) was azidinated (its amino groups wereconverted to azide groups) in 0.5M carbonate/bicarbonate buffer (pH 9.8) ata protein concentration ∼20 mg/ml with 1.2 M excess of the azo transferreagent, imidazole-1-sulfonyl azide hydrochloride [Note: This reagentmust be treated with extreme care. There has been one report of a seriousexplosion during its preparation alternates (35, 36).], and 2 mM CuSO4 ascatalyst for 9 h with stirring on magnetic stirrer. The solution was dilutedwith 50 ml water containing 0.1% Tween-20 (to prevent protein aggre-gation) and washed by ultradialysis using a tangential flow membrane(Pellicon XL 50; 10-kDa cutoff) with EDTA to remove copper, then with0.15 M NaCl 100 mM 4-methylmorpholine, and concentrated to 18 mg/ml.

The protein solution was transferred to a 4 ml Kimball vial, and 5.9 mgtrisaccharide (26 molar equivalents) was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (16 mg).The progress of conjugation was monitored by TLC for consumptionof trisaccharide. After ∼5 h, another portion of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (8 mg) was added, and thepH was adjusted to .8.0 by addition of 4-methylmorpholine. The reactionwas complete when only trace amounts of unconjugated trisaccharide weredetectable by TLC. The conjugate was dialyzed against 20 mM Tris HCl(pH 8.5) and loaded on a DEAE Sepharose CL column equilibrated withthe same buffer. Conjugate was eluted with 0.1 M Tris HCl (pH 7.2) and 1M NaCl. This step was performed to remove Tween-20 present in theconjugate. The conjugate was fractionated on Superdex S-200 column(2 3 100 cm, PBS), and material corresponding to a monomeric fractionwas collected, concentrated, and divided into two equal portions. The firstportion constituted the reference vaccine, TS-TT.

The second portion of TS-TT (10 mg protein) was conjugated withpropargylated laminarin. Briefly, propargylated laminarin (12.5 mg) wasdissolved in 0.2 M Tris HCl buffer (pH 8.0) containing conjugate (10 mgby protein content) in a 4 ml Kimball vial; copper powder (∼20 mg) andisobutanol (50 ml) were added. The vial was closed with a septa andpurged with argon. Reaction was started by addition of bathophenantro-line/Cu+1 catalyst (25 ml per 1 ml reaction mixture) (37). After conjuga-tion, the reaction mixture was filtered, dialyzed against PBS, and purifiedon a Superdex S-200 column. This tricomponent conjugate vaccine, TS-TTwith covalently attached laminarin, was designated TS–TT–laminarin (TS-TT-Lam) vaccine.

The azide groups of both conjugates (TS-TT-Lam and TS-TT) werereduced back to amines by reaction with trimethylphosphine. Conjugates(10 mg/ml) in 0.5 M sodium carbonate were reacted with trimethylphos-phine (50 ml of a 1 M solution in tetrahydrofuran) in 4-ml Kimball glassvials closed with a septa at room temperature, 18 h. Conjugates were thendialyzed against PBS, concentrated, and sterile filtered through 0.22-mmfilters to yield solutions of TS-TT-Lam (2.7 mg/ml) and TS-TT (4.1 mg/ml).

Labeling conjugates with Alexa Fluor 546. To the solutions of the tetanustoxoid conjugate vaccines in PBS (TS-TT and TS-TT-Lam) containing 400mg protein, 1/10 vol 1 M sodium bicarbonate solution was added, followedby Alexa Fluor 546 NHS ester (Molecular Probes, Life Technologies,Grand Island, NY) (20 mg) dissolved in DMSO (∼30 ml). The tubes were

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wrapped in aluminum foil and left on an inverting mixer for 18 h. Puri-fication was performed on PD-10 desalting columns (GE Healthcare)equilibrated with PBS. Fractions containing labeled conjugates were col-lected and concentrated on Amicon Ultra-4 Centrifugal Filter Units (10kDa) to a final concentration of 2.5 mg/ml.

Phosphorylation of curdlan. Curdlan (from Alcaligenes faecalis; Sigma-Aldrich) (1 G), urea (18 G), and formamide (50 ml) were placed in a 500-ml round-bottom flask with magnetic stirring bar mounted in a temper-ature-controlled oil bath. The mixture was first heated to 130˚C to aiddissolution of curdlan. After complete dissolution, the temperature wasreduced to 100˚C. Phosphoric acid (85%, 4 ml) was added in small por-tions over a 2-h period to avoid excessive foaming while maintaining rapidmixing. The reaction mixture was heated for 6 h after addition of the lastportion of phosphoric acid. To the cooled mixture an equal volume ofwater was added and the solution was transferred to a dialysis bag. Dialysiswas first performed against running tap water and then against milliQwater. A small amount of insoluble material was removed by centrifuga-tion (20 min, 10,000 3 g), and purified phospho-curdlan (P-curdlan) waslyophilized.

These b-glucans were solubilized in sterile PBS or RPMI 1640 medium(Life Technologies, Grand Island, NY), sterile filtered, and stored as 10mg/ml stock solutions until use. All b-glucan preparations were endotoxinfree and were used at a working concentration of 100 mg/ml, unless oth-erwise stated.

Cell culture and constructs

The mouse macrophage cell line RAW 264.7 cells (ref. TIB-71; AmericanType Culture Collection, Manassas, VA) were maintained in a-MEM (LifeTechnologies) with 10% heat-inactivated serum (Wisent Bioproducts,St. Bruno, QC, Canada). To generate RAW 264.7 cells stably expressinghuman Dectin-1, the open reading frame of human Dectin-1/CLEC7A(GenBank NM_197947) was subcloned into the pFB-Neo retroviral vector(Stratagene, Agilent Technologies, Santa Clara, CA) containing theneomycin-resistance gene (neor) for selection by G418. The pFB-neoDectin-1 vector was transfected together with the pVPack-GP andpVPack-VSV-G vectors in HEK 293T cells (ATCC CRL-11268), allowingthe formation and secretion of nonreproductive Moloney murine leukemiavirus that is next incubated with RAW 264.7 cells for 24 h before additionof growing media containing 1 mg/ml G418. After 2 wk of selection,Dectin-1 expression was confirmed by immunofluorescence and Westernblot analysis and were further designated as RAW Dectin-1 cells.

Generation of bone marrow–derived DCs

Bone marrow–derived DCs (BMDCs) were prepared from bone marrowcollected from wild-type (WT) C57BL/6 mice maintained at the Faculty ofMedicine and Dentistry animal facility, University of Alberta (Edmonton,AB, Canada). BMDCs were prepared as previously described (38), withminor modifications. Briefly, bone marrow cells (BMCs) were harvestedfrom femurs and tibias of normal C57BL/6 mice and washed with PBS. Togenerate DCs, BMCs were resuspended in complete medium: RPMI 1640(Life Technologies) supplemented with 10% heat-inactivated FCS (WisentBioproducts), 100 U/ml penicillin (Life Technologies), 100 mg/ml strep-tomycin, 10 mM HEPES (Life Technologies), 0.1 mM nonessential aminoacids (Life Technologies), 1 mM sodium pyruvate (Life Technologies),50 mM 2-ME (BioShop Canada), and 20% v/v supernatant from Chinesehamster ovary cells transfected with the plasmid pCDNA3 containing themouse rGM-CSF gene (transduced Chinese hamster ovary cells secretingmouse rGM-CSF were provided by K. Kane, Department of MedicalMicrobiology and Immunology, University of Alberta, Edmonton, AB,Canada). Cell suspension (10 ml) containing 2 3 106 BMCs was culturedin 100-mm–diameter dishes (day 0) and incubated for 10 d at 37˚C ina humidified 5% (v/v) CO2 atmosphere. During the incubation period, cellswere fed with fresh medium containing 20% GM-CSF supernatant on days3 and 5. A total of 10 ng/ml mouse rIL-4 (eBioSciences) was added to themedium on day 6. On day 10 of culture, suspension and loosely adherentcells (immature DCs) were harvested and resuspended in complete me-dium containing 10% (v/v) mouse rGM-CSF supernatant. Cell suspensionwas used for in vitro phenotypic analysis of immature DCs and for co-culture for an additional 24 h with vaccine conjugates, or LPS.

Flow cytometry analysis of BMDC marker expression

On day 10 of culture, analysis of surface marker expression (surfacephenotype analysis) on immature DCs was performed before stimulationwith ligands. Briefly, cells were pelleted by centrifugation and resuspendedat a concentration of 23 107 cells/ml in cold FACS buffer (PBS containing2% FCS). Cell suspensions were then incubated on ice for 10 min withanti-FcRcII/III Ab (anti-mouse CD16/32; eBioscience) for FcR blocking.

Aliquots of 1 3 106 cells in 50 ml were then stained with a master mix offluorochrome-conjugated Abs to stain for the various DC markers de-scribed in Table I, or with a mix of corresponding IgG isotype controls.Abs used were from eBiosciences (Table I) and are as follows: PE-Cy7–conjugated anti-CD11c, FITC-conjugated anti-CD86 (B7-1), FITC-conjugatedanti-F4/80, PE-conjugated anti-CD80 (B7-2), and eFluor 450–conjugated anti-MHC II (I-A/I-E). Isotype controls were done with similarly conjugatedIgG. One aliquot of cells was left unstained for use as a control. Afterincubation with the Ab mix for 30–40 min at 4˚C in the dark, cells werewashed twice and resuspended in cold FACS buffer, and samples wereacquired on a LSR II cytometer (BD Biosciences, San Jose, CA).Compensation was performed before each data collection using BDBiosciences CompBeads anti-rat/hamster Ig, k/negative control com-pensation particles set, according to manufacturer’s recommendations.Gating was performed for elimination of dead cells. Software analysis offlow cytometric data was performed on FSC3 Express from De Novo soft-ware (Los Angeles, CA).

Cytokine quantification in culture supernatants using ELISAs

For analysis of cytokine production, immature BMDCs were harvested onday 10 of culture and resuspended in complete RPMI 1640 mediumcontaining 10% (v/v) mouse rGM-CSF supernatant. Cells were plated at1 3 106 cells/well in a 12-well plate and stimulated with 100 mg/mlvaccine conjugates (TS-TT or TS-TT-Lam conjugates), or with 1 mg/mlLPS. Cell culture supernatants were collected after 24-h incubation.Concentration of the cytokines IL-4, IL-6, IL-12p70, TNF-a, and TGF-b1in supernatant was assayed by precoated sandwich ELISA kits (R&DSystems, Minneapolis, MN). All of the samples were measured in tripli-cate, according to the manufacturer’s instructions. Statistical analysis wasperformed with Prism software using Student t test (GraphPad Software).

Immunofluorescence and Western blotting

Phosphorylated Syk was analyzed by immunofluorescence or by immu-noblotting after 10-min stimulation at 37˚C with 100 mg/ml vaccine con-jugates or P-curdlan. Src activation was detected by immunofluorescenceafter 5-min stimulation. For immunofluorescence, cells were grown over-night on coverslips at 40% confluency, and the following day cells wereserum starved for 4–5 h in serum-free a-MEM prior to stimulation. Afterstimulation, cells were washed three times with ice-cold PBS, fixed on icein 4% paraformaldehyde, and blocked in PBS containing 3% BSA plus 3%fish skin gelatin (Sigma-Aldrich). To stain for surface dectin-1, cells wereincubated before permeabilization with mouse anti-human Dectin-1, fol-lowed by a dye-coupled anti-mouse secondary Ab. Next, cells were per-meabilized with 0.1% (v/v) Triton X-100 in PBS, and stained for 30 minwith either rabbit anti-phospho Syk (Tyr352) or rabbit anti-phospho Src(Tyr418) conjugated to Alexa Fluor 488. Cells were then washed and in-cubated with Alexa Fluor 488–conjugated goat anti-rabbit IgG (Invi-trogen), and DAPI was used to highlight cell nuclei. Following staining,coverslips were washed, mounted with DAKO mounting medium (DAKO,Agilent Technologies, Santa Clara, CA) and viewed on a spinning-diskconfocal microscope (WaveFx from Quorum Technologies, Guelph, ON,Canada).

SDS-PAGE and immunoblotting

For analysis of Syk phosphorylation by immunoblotting, cells were culturedovernight in 6-well plates. The following day, cells at 80% confluency wereserum starved for 5 h prior to stimulation. After incubation at 37˚C with 100mg/ml P-curdlan or vaccine conjugates for 10 min, cells were chilled onice, washed with cold PBS, and lysed by addition of phosphorylation lysisbuffer (20 mM MOPS, 1% Triton X-100, 2 mM EGTA, 5 mM EDTA, 1mM sodium orthovanadate, 2 mM EGTA, 5 mM EDTA [pH 7.0]) con-taining phosphatase inhibitor mixture (PhosSTOP, Roche Applied Science,Laval, QC, Canada) and protease inhibitor mixture (Sigma-Aldrich). Cellswere scraped and cell debris were removed by centrifugation at 14,000rpm for 20 min at 4˚C. The protein content of lysates was quantified usingthe Bio-Rad bicinchoninic acid protein assay. For Western blot analysis,cleared lysates were resolved by SDS-PAGE, transferred to nitrocellulosemembrane, and immunoblotted with the following Abs: rabbit anti-phospho Syk (Tyr525/526), rabbit anti-phospho Syk (Tyr352), rabbit anti-Syk, or mouse anti-actin for loading control. This was followed by incu-bation with appropriate HRP-conjugated secondary Abs and detection byan ECL kit from Roche.

NF-kB nuclear localization

RAW-Dectin-1 cells were plated overnight onto glass coverslips in a 12-wellplate. The following day, cells were stimulated at 37˚C for 10 min with

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vaccine conjugates or ligands in HEPES containing RPMI 1640 medium(Wisent Bioproducts). After stimulation, cells were washed three timeswith PBS, and fresh media was added for another 20 min to allow NF-kBnuclear translocation. Cells were then fixed with 4% paraformaldehyde,blocked, and permeabilized with PBS containing 0.5% Triton X-100. Cellswere stained with anti-p65 Ab, followed with Alexa Fluor 488–conjugatedgoat anti-mouse IgG (Molecular Probes), and DAPI was added to detectcell nuclei. Following staining, coverslips were washed and mounted, andp65 nuclear localization was examined by confocal microscopy.

Dectin-1 ligand-binding assay

RAW 264.7, referred to as RAW WT and RAW Dectin-1 cells, were platedovernight on glass coverslips in a 12-well plate. The following day, cells at60% confluency were incubated on ice for 10 min with Alexa Fluor 546–labeled vaccine conjugates. Cells were then washed four times with ice-cold PBS to remove unbound particles, fixed with 4% paraformaldehyde,and blocked with PBS containing 3% BSA plus 3% fish skin gelatin(Sigma-Aldrich). For detection of surface Dectin-1 on the plasma mem-brane, nonpermeabilized cells were incubated with mouse anti-humanDectin-1 Abs, followed by incubation with Alexa Fluor 488–conjugatedanti-mouse IgG and DAPI. Coverslips were mounted with DAKO andexamined using confocal microscopy.

Confocal microscopy

Confocal microscopy was performed on a spinning disk microscope(WaveFx from Quorum Technologies, Guelph, ON, Canada) set up on anOlympus IX-81 inverted stand (Olympus Canada, Richmond Hill, ON,Canada). Images were acquired through a 360 objective (N.A. 1.42) withan electron multiplying charge-coupled device camera (Hamamatsu, Ja-pan). Z-slices of 0.3 mm were acquired through the cells using Volocity(Improvision, Perkin-Elmer, Waltham, MA).

Mice vaccination

The effect of vaccination with the TS-TT-Lam conjugate was studied inthree mouse strains, as follows: BALB/c, C57BL/6, and CD1 (outbredstrain). Groups of 10 mice from each strain were immunized with TS-TTconjugates. Control groups (10 mice each) were given the same conjugatethat lacks the laminarin substituent. Both Ags were given as emulsion inmineral oil (IFA). Each dose contained 20 mg conjugate (equivalent of 1.25mg trisaccharide) in 300 ml to the formulation administered i.p. (200 ml)and s.c. (100 ml). C57BL/6 and CD1 mice were immunized four times at3-wk intervals, and BALB/c mice received four injections at 2-wk inter-vals. Sera were collected 10 d after each immunization. Ten days after thefourth immunization, mice were bled and exsanguinated.

b-mannan–specific Ab titration

Polystyrene 96-well plates were coated overnight with trisaccharide-BSAconjugate (11) at a concentration of 5 mg/ml in PBS or with C. albicansphosphomannan complex (39) at the same concentration in 0.05 M car-bonate buffer (pH 9.8). After washing with PBS containing 0.1% Tween-20 (PBST), 100 ml serial √10 dilutions of sera (starting from 1023) wereadded to each well. A solution of 0.1% skim milk (Difco) in PBST wasused for dilutions to prevent nonspecific binding. Plates were sealed andincubated for 2 h at room temperature. After washing with PBST, a re-porter Ab, anti-mouse IgG, HRP conjugate from Kirkegaard & PerryLaboratories, in 0.1% skim-milk PBST, at a dilution 1/2000, was appliedand plates were incubated for 1 h at room temperature. Plates were washedagain with PBST and color developed with HRP substrate system (Kir-kegaard & Perry Laboratories) for 15 min. The reaction was stopped with1 M phosphoric acid, and absorbance was measured. End point titres wererecorded for dilutions that gave an OD reading of 0.2, which correspondedto an OD at least 0.1 U above background.

Ab isotyping and titration

ELISA plates were coated with a trisaccharide-BSA conjugate, as describedabove. Sera dilutions were chosen to yield optimal OD signal based on thetotal IgG titration. Subclasses were detected for duplicate samples witha subisotyping kit (Bio-Rad) and expressed as a percentage of a sum of ODsignals read for individual subclasses. The change in subclass distributionwas analyzed for statistical significance. A frequency of each four Absubclasses was treated as a point on a simplex; the Bhattacharyya measurewas used to compare two distributions (40). If pðiÞ and p9ðiÞ represent

frequencies of i ¼ 1; 2; 3; 4 subclass and +4

i¼1pðiÞ ¼ +4

i¼1p9ðiÞ ¼ 1, the

Bhattacharyya metric is defined as Bhðp; p9Þ ¼ +4i¼1

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffipðiÞp9ðiÞp

. Dis-tribution of this statistic was estimated with permutation procedure. Dif-

ferences between two vaccines were considered at a = 0.05. Differencesbetween effect sizes of two mouse strains were tested with Mahalanobisdistance estimated based on bootstrap samples. Means’ SEs were com-puted with bootstrap approach.

Statistical analysis of sera titers

To test differences between two response curves after three vaccinationswith TS-TT-Lam and TS-TT, the least squares method was used to derivea best-fitting line for each mouse. Thus, the set of vectors of three boostmeasurements Xij = b1ij,b2ij,b3ij, where b1ij was the first boost of the jthmouse in the ith group and so on, was replaced with a set of vectors Yij =(aij,aij), where aij and aij were the intercept and slope of the regressionline for the jth mouse in the ith treatment group. Next, Hotellings’s T2

statistic was calculated to determine whether the responses after vaccina-tion with two vaccines were similar. In the case of multivariate ANOVA,Pillai-Bartlett statistic was computed. Because of small samples, sizepermutation tests were applied. Means, SD, and SEMs were calculated.Cohen’s d was used as the measure of the effect size. All data were ana-lyzed with R platform version 2.11.1. Results inferior to a = 0.05 wereconsidered statistically significant. Outliers were detected with Grubb’stest.

ResultsWe generated a tetanus-toxoid–bearing b-mannan vaccine, as in-itially described in Lipinski et al. (37) and conjugated additionallaminarin molecules (Fig. 1). Laminarin is a b-glucan recognizedby Dectin-1 (34, 41), a surface receptor that is highly expressed inDCs and macrophages. Tetanus toxoid was reacted with the diazotransfer reagent, imidazole-1-sulfonyl azide hydrochloride, toconvert amino groups into azide groups. Transformation of aminesinto azides prevented cross-linking of the protein following acti-vation of aspartic and glutamine side chain carboxylic acids forcoupling with the amino-terminated trisaccharide hapten. Theresulting conjugate was divided into two portions; one was con-jugated to propargylated laminarin using click chemistry, and theother was used as the control vaccine. The residual azide groups ineach conjugate were reduced back to the original amino forms togive two conjugates designated TS-TT and TS-TT-Lam (Fig. 1).We first decided to validate the Dectin-1–specific targeting of

this trifunctional vaccine. We generated a stable murine macro-phage cell line expressing Dectin-1 at noticeable level at the cellsurface by viral transduction of RAW 264.7 cells (called RAWDectin-1 thereafter) and neomycin selection. As shown in Fig. 2A,the human Dectin-1 A isoform is readily detectable at the cellsurface upon immunostaining of nonpermeabilized cells. Simi-larly, immunoblotting (Fig. 2B) of whole-cell lysates preparedfrom cells expressing Dectin-1 demonstrates the b-glucan receptorexpression at a molecular mass of ∼40–50 kDa. This m.w. cor-responds to the complex glycosylated form of the transmembraneprotein confirming the appropriate folding, targeting, and surfaceexpression of Dectin-1 in the RAW macrophages.We next investigated whether the presence of the laminarin

polysaccharide on the tetanus toxoid (TS-TT-Lam) would allowfor a better binding of the vaccine by macrophages expressingDectin-1 compared with the TS-TT compound (tetanus toxoid withb-mannan only). To visualize the binding of the vaccine conjugateto RAW Dectin-1 cells, these compounds were coupled to AF546(see Materials and Methods). Binding was achieved by incubationof the RAW Dectin-1 cells with the fluorescent compounds, TS-TT-AF546 or TS-TT-Lam-AF546, for 10 min, followed by washeswith PBS and fixation with 4% paraformaldehyde (PFA). Dectin-1immunostaining was performed in parallel, and, as shown in Fig.3A, only the vaccine coupled to laminarin was able to bind RAWDectin-1 cells. We confirmed that the binding of the TS-TT-Lam-AF546 was specific to the cells expressing Dectin-1, as RAW WTwere not capable of recognizing the laminarin conjugate (data notshown).

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Dectin-1 signaling requires phosphorylation of its cytosol hem-ITAM motif on tyrosine 15 by a Src family kinase (SFK), allowingthe phospho-ITAM–like domain to recruit the Syk to mediate

further downstream signaling (39, 41). To verify that TS-TT-Lamglycoconjugate was indeed able to activate SFK, we tested theappearance and colocalization of the active form of SFK with

FIGURE 1. Conjugation scheme for synthesis of the two conjugates, TS-TT and TS-TT-Lam.

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Dectin-1 following binding of fluorescent TS-TT-Lam. Followingstimulation with TS-TT-AF546 or TS-TT-Lam-AF546, cells werefixed, permeabilized, and immunostained with an anti-phospho(Y418) Src Ab that reports SFK activation. Cells stimulatedwith TS-TT compound did not show significant staining for thephosphorylated form of Src (Fig. 3B). In contrast, the laminarin-carrying vaccine showed a pronounced increase in phosphory-lated SFK (Fig. 3B).We next confirmed the recruitment and activation of Syk by

immunostaining and immunoblotting (Fig. 3C, 3E). Cells incu-bated with TS-TT or TS-TT-Lam for 10 min were either fixed andimmunostained for Dectin-1 and phospho-Syk or lysed and ana-lyzed by Western blotting with anti-phospho Syk Abs. By im-munostaining (Fig. 3C), activation of Syk could be detected onlyupon stimulation with TS-TT-Lam, but not with TS-TT. This re-sult was confirmed by the Western blot experiment showing that

TS-TT-Lam activates Syk in a Dectin-1–dependent manner. InRAW cells that do not express Dectin-1, addition of either TS-TTor TS-TT-Lam did not increase Syk phosphorylation on eitherY525/526 or Y352 (Fig. 3E). Similarly, Syk was not activated inthe Dectin-1–expressing cells upon addition of TS-TT. However,Syk phosphorylation of Y525/526 and Y352 could only bedetected when the laminarin conjugate was used on Dectin-1–expressing cells (Fig. 3E). This result indicates that, followingbinding to the b-glucan receptor, the oligovalent TS-TT-Lammolecules are capable of triggering Dectin-1 activation and, inparticular, the Syk pathways. These results confirm that thelaminarin conjugate is able to activate SFK and the Syk adaptorproteins, which are two crucial effectors of the Dectin-1 signalingpathway. Finally, we tested the activation and translocation of theNF-kB in fixed RAW Dectin-1 macrophages by immunostainingfor the p65 subunit of NF-kB in parallel with DAPI following 10min of incubation with either TS-TT or TS-TT-Lam. Althoughnuclear translocation of NF-kB was not seen in cells treated withTS-TT, TS-TT-Lam led to its pronounced translocation (Fig. 3D).Activation of NF-kB by the laminarin-containing vaccine furtherillustrates the capacity of this compound to stimulate innate im-mune cells in a Dectin-1–dependent fashion.Using macrophages stably expressing Dectin-1 at the cell surface,

we have validated that the laminarin-containing vaccine, TS-TT-Lam, was able to specifically interact with the b-glucan receptorand activate well-characterized downstream effectors. Before test-ing the vaccines directly in animals and to better define the immuneresponse triggered by these compounds, we examined in vitro ac-tivity of the TS-TT and TS-TT-Lam conjugates.Dectin-1 is expressed on cells from the monocytic lineage,

especially on DCs (42), which are responsible for promoting an-tifungal immunity during pathogenic challenges (18, 20). To de-termine the potential of TS-TT-Lam conjugates in stimulating im-mune responses, we analyzed the response of primary BMDCs tob-glucans and the vaccine conjugates.Preparation of DCs was performed from bone marrow lavages

of C57BL/6 mice and differentiation in presence of GM-CSF andIL-4. DC surface marker characterization was performed by flowcytometry (Fig. 4A) with a series of Abs described in Table I. Asexpected for BMDCs, the majority of cells showed expression ofCD11b and CD11c surface proteins. A small proportion (∼9%) ofthe culture expressed the macrophage marker F4/80, but the ma-jority of the cells presented an immature DC phenotype witha moderate expression of MHC-II and CD86 and high expressionof CD80. Additionally, Dectin-1 immunofluorescence stainingfurther confirmed the expression of the b-glucan receptor in theseimmature BMDCs. Cells plated on glass coverslips were fixed andnonpermeabilized before addition of a rat anti-mouse Dectin-1 Ab(Fig. 4B). The expression of Dectin-1 in primary dendritic hasbeen shown to promote several cell responses upon binding ofparticulate b-glucans (18, 43). We performed a series of experi-ments to test the responsiveness of immature DCs to Dectin-1ligands. BMDCs were incubated for 10 min with P-curdlan, andactivation of Syk and nuclear translocation of NF-kB were ex-amined. Using the anti-phospho Syk Ab, we verified that b-glu-cans trigger Syk activation immediately following incubation withthe ligand. Incubation of BMDCs with P-curdlan for 10 minresulted in the appearance and recruitment of a phosphorylatedform of the kinase, as shown by the strong near-membrane lo-calization of phospho-Syk (as shown in the middle panel of Fig.4B). NF-kB translocation was analyzed after 10 min of ligandtreatment, followed by another 20 min of incubation. Immuno-fluorescence staining of NF-kB in BMDCs untreated or treatedwith a soluble b-glucan demonstrated the responsiveness of DCs

FIGURE 2. Characterization of RAW Dectin-1 cells. Macrophages

RAW 264.7 were virally transduced with the pFB-neo plasmid (Stratagene,

Agilent Technologies, Santa Clara, CA) containing the human Dectin-1 A

cDNA. Cells were selected using 1 mg/ml G418 in the culture medium.

Expression and surface targeting of Dectin-1 in the RAW Dectin-1 cells

were assessed. (A) Immunofluorescence staining on nonpermeabilized

cells (surface Dectin-1) with a mouse anti-Dectin-1 Ab, followed by

a donkey anti-mouse coupled to Cy3, revealed the membrane targeting of

the receptor. Following permeabilization with 0.1% Triton X-100, the

same cells were stained with the same Ab, except that the secondary was

coupled to Alexa Fluor 488, revealing internal structures. DAPI was used

to localize the cells’ nuclei. Data are representative of five independent

experiments. Scale bar, 2 mm. (B) Western blotting of whole-cell lysate

prepared from RAW WT- and RAW Dectin-1–expressing cells. For im-

munoblotting, the goat anti–Dectin-1 Ab (R&D Systems) was used at

1:1000, followed by an anti-goat IgG coupled to HRP (1:5000). In the

RAW Dectin-1 lane, several bands appeared upon ECL treatment and film

development. The upper and large bands correspond to the complex gly-

cosylated form of the receptor, whereas the smaller one (30 kDa) corre-

sponds to the core glycosylated (determined by PNGase and EndoH

treatment; data not shown).

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to Dectin-1 ligand (Fig. 4C). In unstimulated cells, NF-kB isdispersed throughout the cytosol, whereas cells incubated withb-glucans showed a strong relocalization to the nucleus, asdemonstrated by the DNA-binding fluorophore DAPI (Fig. 4C).We have therefore shown that immature DCs express Dectin-1,and they respond to treatment with pure b-glucan (P-curdlan) bytriggering Syk activation and NF-kB translocation in the nucleus.We next tested the action of our vaccine conjugates on primary

DCs. Secretion of specific cytokines and appearance of surfacemarkers specific to DCs maturation were examined (Fig. 5). Im-mature BMDCs were treated with TS-TT, TS-TT-Lam, or LPS for24 h before they were harvested. Cell supernatant was used for thedetermination of the secreted cytokines by specific ELISAs, andthe cells were gently scraped, stained, and analyzed by flowcytometry for markers of maturation such as MHC-II and CD86.Fig. 5A shows representative traces for the expression of CD86and MHC-II and the quantification of the higher intensity section(M1) of the signals indicative of the upregulation of thesereceptors during maturation. LPS is by far the strongest inducer ofBMDC maturation, as it promoted a significant increase in theexpression level of CD86 and MHC-II after 1 d compared withcontrol or vaccine treatment (Fig. 5A). Stimulation with the vac-cine conjugates for the same period had milder effects on theupregulation of the differentiation markers. TS-TT induceda small increase in the percentage of cells expressing high levelsof MHC-II compared with WT (21.72 6 2.28 versus 16.37 60.17, p . 0.05) and of CD86 (24.08 6 0.81 versus 20.17 6 0.93,p # 0.05). By contrast, TS-TT-Lam induced a more pronounced

induction, as supported by observations of a significantly greaterfraction of cells expressing these markers in treated cells com-pared with unstimulated (MHC-II: 28.79 6 1.31 for TS-TT-Lam versus 16.37 6 0.17 for unstimulated, p # 0.01, andCD86: 28.55 6 0.59 for TS-TT-Lam versus 20.17 6 0.93 forunstimulated, p # 0.001). Overall, these results indicate that thevaccine conjugates have the capacity to induce the differentiationof immature DCs.We next looked at the secretion of several cytokines in the culture

medium recovered from BMDCs after 24 h of stimulation with TS-TT, TS-TT-Lam, or LPS. Fig. 5B shows the ELISA quantificationof the following five cytokines: IL-4, IL-12, TNF-a, TGF-b, andIL-6. Stimulation with the TLR4 ligand LPS led to secretion oflarger amount of cytokines, especially of IL-4, IL-12, TNF-a, andIL-6. Although TS-TT conjugate vaccine did not result in highercytokine production when compared with untreated cells, TS-TT-Lam augmented the secretion of IL-4, IL-6, and TGF-b. Insummary, we show that conjugating b-glucans to the TS-TTvaccine boosted its effects and especially raised the secretion ofIL-4, IL-6, and TGF-b. Taken together, our results to date con-firmed that TS-TT-Lam binds to and activates the b-glucan re-ceptor in RAW macrophages expressing Dectin-1 and also inBMDCs. In the primary cells, this activation promotes the dif-ferentiation of DC and more significantly induces the productionand secretion of cytokines that will act to stimulate the immunesystem.Validation of the immunization potential of the TS-TT-Lam vac-

cine was next carried out in animal model. Groups of 10 mice from

FIGURE 3. RAW Dectin-1 macrophages respond to

laminarin-containing vaccine. (A) RAW Dectin-1 cells

were incubated on ice for 10 min with fluorescently

labeled vaccine conjugates (TS-TT-AF546, top row or

TS-TT-Lam-AF546, lower row), followed by parafor-

maldehyde fixation. Dectin-1 was immunostained in

nonpermeabilized macrophages with the mouse anti–

Dectin-1 and AF488-conjugated anti-mouse secondary

Abs. Activation of Dectin-1 downstream effectors is

shown in (B)–(E). RAW Dectin-1 macrophages were

incubated with TS-TT-AF546 or TS-TT-Lam-AF546

for 10 min at 37˚C before fixation with 4% PFA. (B)

Activation of SFK was visualized using rabbit anti-

phospho Src (Y418) coupled to AF488 (Invitrogen). (C)

Syk recruitment and activation were observed using

a rabbit anti-phospho Syk (Tyr352) and an anti-rabbit

AF488. (D) NF-kB translocation to the nucleus was

determined upon an additional 20-min incubation of

the cells following ligand incubation. Cells were fixed

and permeabilized, and NF-kB was labeled using the

rabbit anti-p65 Ab (Santa Cruz Biotechnology), fol-

lowed by an anti-rabbit AF488 (green) Ab in parallel

to DAPI (blue). (E) RAW Dectin-1 or RAW WT cells

were treated with TS-TT or TS-TT-Lam for 10 min at

37˚C, and protein lysates were prepared, separated by

SDS-PAGE, transferred on nitrocellulose membrane,

and blotted with Abs against phospho-(Y352) or

phospho-(Y525/Y526) Syk and against total Syk and

b-actin as loading controls. Data are representative of

three similar experiments. Scale bars, 2 mm.

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three different mouse strains (BALB/c, C57BL/6, and outbred CD1)were vaccinated with either TS-TT-Lam or TS-TT conjugates fourtimes (prime injection and three boosts). Serum samples were taken10 d after each booster injection and analyzed using ELISA for titers

against the trisaccharide hapten attached to a heterologous carrierprotein (TS-BSA) and against C. albicans cell wall phosphomannan.Results of the sera titers for the three mouse strains after each boostare shown on Fig. 6A and demonstrate that Ab titers increased

FIGURE 4. BMDCs respond to Dectin-1 ligands.

(A) Flow cytometry profile confirming the pheno-

type of the immature BMDCs (histograms in black

indicate isotype controls). More than 80% of

CD11b+/CD11c+ also express CD80. The moderate

expression of CD86 and MHC-II indicates that

these DCs are immature. The low expression of the

macrophage marker F4/80 indicates that the cell

populations consist mainly of DCs. (B) Syk acti-

vation following BMDC treatment with soluble

b-glucan, P-curdlan, or vehicle control (unstimu-

lated). Cells were incubated for 10 min at 37˚C

with or without the Dectin-1 ligand and fixed with

PFA. Dectin-1 was immunostained first on non-

permeabilized cells with a rat anti-mouse Dectin-1

Ab and a Cy3-conjugated anti-rat Ab (red). Phos-

pho-Syk immunostaining was performed after per-

meabilization with 0.1% Triton X-100 using

a rabbit anti-phospho Syk and AF488-conjugated

secondary Ab. (C) NF-kB translocation in BMDCs

stimulated or not with P-curdlan. Following incu-

bation without or with 100 mg/ml P-curdlan,

BMDCs were washed and incubated at 37˚C for

another 20 min, after which they were processed for

immunostaining for NF-kB, as described in Mate-

rials and Methods. Data in (A) are representative of

three independent experiments, and data in (B) and

(C) are representative of five experiments. Scale

bars, 2 mm.

Table I. List of Ags for the characterization of BMDCs used for flow cytometry

Ag CD11c CD11b CD80 (B7-1) CD86 (B7-2) F4/80 MHC-II

Fluorophore PE-Cy7 Alexa Fluor 700 PE FITC FITC eFluor 450Final dilutions 1/1000 1/500 1/1000 1/800 1/500 1/1000Isotype controls Hamster IgG Rat IgG2b, k Rat IgG2a, k Rat IgG2b, k Rat IgG2b, k Rat IgG2b, k

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following each challenge. Compared with TS-TT–vaccinated mice,TS-TT-Lam conjugate vaccination resulted in 5- to 10-fold highertiters against both synthetic Ag and cell wall phosphomannan (me-dian values are indicated on the graph) in all tested mice strains (Fig.6B). Because the vaccine was derived from a single batch, eachconjugate had exactly the same number of trisaccharide epitopes. AbIgG subisotype distribution (Fig. 6C) was also influenced by theconjugate used during the vaccination protocol. Vaccination withTS-TT-Lam resulted in an increased level of IgG1 (∼50%) anddecrease of other subclasses, particularly IgG2a, in BALB/c andC57BL/6 strains. The changes in subclass distribution were signif-icant in both BALB/c and C57BL/6 strains (p = 0.04676 and0.00375, respectively), but the results in CD1 mice were inconclu-sive due possibly to the low number of animals and high variationbetween individual mice.In summary, we show that the novel laminarin-bearing vaccine is

recognized by and activates Dectin-1 cells. In the case of primaryDCs, this activation leads to the production of several regulatorycytokines, such as IL-4, IL-6, and TGF-b. Finally, animals chal-lenged with TS-TT-Lam showed increased Ab responses, in the

form of IgG isotypes, directed against the b-mannan trisaccharideand the tetanus toxoid compared with the TS-TT conjugate.

DiscussionWe have previously shown that five of six synthetic b-mannotriose–peptide conjugates conferred good to excellent protection againstC. albicans challenge if the glycopeptides were administered viaAg-pulsed DC vaccination (44). This was in sharp contrast to thepoor immunogenicity of b-mannotriose–tetanus toxoid conjugatein mice (32, 33, 37). These observations prompted us to considerDC targeting as a means to enhance the antigenicity of theb-mannan–tetanus toxoid conjugate. Dectin-1 Ab-mediated tar-geting of the protein Ag OVA to DC has been reported to yieldsuperior response to the protein Ag OVA, whereas targeting viaDEC205 did not (45). In this study, we elected to target DCs byattaching b-glucan—a ligand for the C-type lectin receptor Dectin-1—to the b-mannan–tetanus toxoid conjugate. This approach hasthe potential for a dual outcome: by elevating the response to theb-mannan of C. albicans while taking advantage of the proventargeting and antifungal response of b-glucan conjugates.

FIGURE 5. Activation of BMDCs and secreted

cytokine profile upon stimulation with vaccine

conjugates. (A) Flow cytometry analysis of the cell

surface markers expressed on BMDCs treated for

24 h with TS-TT (red), TS-TT-Lam (blue), LPS

(green), or just tissue culture medium (gray).

BMDC activation was quantified (right histograms)

by the increased expression of CD86 (top graphs)

and MHC-II (lower graphs) using the percentage of

cells appearing in the higher intensity level section

of the graphs and marked M1. (B) Cytokine

measurements performed by ELISAs on BMDCs

treated for 24 h. The level of IL-4, IL-12, TNF-a,

IL-6, and TGF-b was determined by comparison

with protein standards provided by the manufac-

turer and as described in Materials and Methods.

Data in (A) and (B) are mean 6 SE of three inde-

pendent experiments. ***p # 0.001, **p # 0.01,

*p # 0.05. ns: p . 0.05, calculated using Student

t test.

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As shown in Fig. 6B, DC targeting of the vaccine resulted in Abtiters that were 5- to 10-fold higher than those observed for a TTconjugate bearing only the b-mannan B cell epitope. The muchhigher Ab levels observed in BALB/c mice were also reflected inthe production of protective b-mannan–specific mAbs, which is insharp contrast to numerous unsuccessful attempts when mice wereimmunized with the TS-TT conjugate (T. Lipinski, X. Wu, J.M.Sadowska, and D.R. Bundle, unpublished observations; see alsoRefs. 32, 33).The role of Dectin-1 in the binding and activation of innate

immune cells was initially confirmed by in vitro experiments ona cell line of macrophages expressing a human isoform of Dectin-1. Stable Dectin-1–expressing cells were obtained after viraltransduction of RAW 264.7 macrophages, and Dectin-1 wasdetected on their cell surface (Fig. 2A). This provided us witha system that allowed testing of ligand binding and activation ofdownstream Dectin-1 effectors in comparison with the WT cells(RAW WT). The laminarin-containing conjugate (TS-TT-Lam)could bind to RAW Dectin-1 cells only, whereas the b-man-nan–only vaccine (TS-TT) did not show any significant inter-action (Fig. 3A).

Laminarin, a small soluble ligand, has been reported to bindDectin-1 (41), but it remains unclear whether the small b-glucancan produce specific Dectin-1–mediated responses. Goodridgeet al. (46) recently reported that soluble b-glucans are also inef-fective in triggering Dectin-1 activation. Because our TS-TT-Lamconjugate carries approximately three laminarin chains per vac-cine molecule, we surmised that this oligovalent presentation, incontrast to the soluble form, could activate Dectin-1 by engaginga series of receptors. If so, this would suggest that even relativelyshort b-glucan sequences might be capable of activating Dectin-1,provided that they are presented in multivalent format. In accor-dance with this notion, we showed in this study that only theb-glucan–containing conjugate (TS-TT-Lam) was able to stimu-late effectors of the Dectin-1 pathway (Fig. 3). Upon ligandbinding, tyrosine 15 phosphorylation of Dectin-1 by Src familykinases enables the recruitment of Syk on the resulting phosphohemITAM motif (18, 24). In RAW Dectin-1 macrophages, TS-TT-Lam was able to do the following: 1) increase phospho-SFK in thevicinity of bound conjugates (Fig. 3B), leading to 2) activation ofSyk (Fig. 3C, 3E) and 3) inducing the translocation of NF-kB tothe cell nucleus (Fig. 3D). Activation of Src family kinases, Syk

FIGURE 6. (A) Statistical analysis of response curves resulting from data for average titer after each booster injection showed that curves for TS-TT-Lam

and TS-TT conjugates are different for BALB/c (p = 0.0066 for BSA-TS, and p = 0.00012 for cell wall) and CD1 (p = 0.0432 for BSA-TS, and p = 0.0346

for cell wall) mice strains. (B) The end point (third boost) analysis included results from C57BL/6 mice and showed that TS-TT-Lam induced higher

response to BSA-TS and cell wall in all tested strains (multivariate ANOVA test, p = 0.0336). Calculated effect size (Cohen’s d) gave d = 0.448 for BSA-TS

and d = 0.525 for cell wall that corresponds to moderate titer increase when vaccinating with TS-TT-Lam conjugate. (C) IgG isotype subclasses analysis in

end point (third boost) mouse sera. Statistical analysis described in Materials and Methods.

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and NF-kB, only occurred in Dectin-1–expressing cells with thelaminarin-containing conjugate demonstrating the specificity ofthis Dectin-1–dependent signaling pathway to b-glucans. Fol-lowing binding of the b-glucan–conjugated vaccine TS-TT-Lam,it is taken up by RAW Dectin-1 macrophages, resulting in stim-ulation of the cells.To further confirm the immune stimulation potential of these

glycoconjugates, we tested their effects on immature BMDCs.Dectin-1 expression and activation by the conjugate vaccine TS-TT-Lam on BMDCs were confirmed (Fig. 4). The poor immu-nogenicity of the TS-TT conjugate can be explained by the in-ability of TS-TT to bind to macrophages or BMDCs, presumablyresulting in poor uptake and processing for presentation by DCs.To validate the potency of TS-TT-Lam in inducing an immuneresponse, we determined its effect on primary innate immunecells. We used BMDCs grown in medium containing IL-4 andGM-CSF. After 10 d of culture, BMDCs were mainly immature,as shown by the following flow cytometry profile: majority ofCD11c+-CD11b+-CD80+ and moderate expression levels of MHC-II and CD86 (Fig. 4A). In response to 24-h incubation with theTS-TT-Lam conjugate, a significant fraction of cells expressedhigher levels of MHC-II and CD86 (Fig. 5A) compared withunstimulated conditions, indicating an induction of the differen-tiation of the DCs toward a more mature phenotype. These resultsare in agreement with reports demonstrating that laminarin andcurdlan can activate immature DCs (47). In addition, curdlan wasshown to induce cytotoxic T cell priming through the stimulationof IFN-g secretion by DCs (48). DCs are immune regulatory cellsthat can produce selective molecules that will influence the overallimmune response (49–51).Cytokines secreted by BMDCs stimulated with the TS-TT-Lam

vaccine showed a modest increase in TNF-a and IL-12. Instead, wemeasured a significant augmentation of IL-4 secretion (p # 0.05)and a stronger induction of TGF-b and IL-6. IL-4 secretion favorsthe differentiation of type 2 Th cells, which in turn promotes Abproduction by B cells and isotype class switching (52). TGF-b isan important regulatory signal for Foxp3+ regulatory T cells (Treg)(53, 54). However, when secreted in conjunction with IL-6, thesecytokines initiate the differentiation of naive T cell into IL-17–secreting T cells called Th17 cells (55–57). In contrast to thepotential induction of Treg by TGF-b, our measurements of in-creased IgG titers following each vaccination boost support thelatter alternative. Although we did not investigate the exact natureof the T cell response triggered in vivo, our results indicate thatTreg cells are not being activated. The concomitant increased se-cretion of TGF-b and IL-6 by BMDCs stimulated by thelaminarin-containing vaccine suggests that Th17 cells are possiblyactivated. In the context of Dectin-1, several groups have reportedthe capacity of DCs to stimulate T cell–mediated immune re-sponses (45, 48) and can in addition activate Th17 cells throughthe secretion of IL-6 and TGF-b (14, 58, 59). This Th17 immuneresponse is promoted by Dectin-1 and leads to a more efficientantifungal immunity through the recruitment of neutrophils to theinfection site (13, 16, 60–62).Compared with bicomponent conjugate vaccines, such as OVA-

laminarin (44) or our previous TS-TT compound (63), the tri-component TS-TT-Lam promotes multiple immune pathways andenables a more robust immune response. Indeed, immunization ofthree strains of mice resulted in an improved vaccination docu-mented in Fig. 6 by higher IgG Ab titers against the C. albicanscell wall b-mannan and the synthetic b-mannan trisaccharide con-jugated to BSA. Whereas carbohydrates are T cell–independentAgs, recent studies of polysaccharide conjugate vaccines demon-strated their capacity to promote T cell–dependent responses with

induction of memory and IgM to IgG Ab class switching (44, 64).The same Ab class switching was induced by the TS-TT-Lam fol-lowing mice immunization, indicating T cell–supported B cell dif-ferentiation and immune memory (Fig. 6C). Taken together, ourresults suggest that at least two distinct immunoregulatory mecha-nisms are stimulated by TS-TT-Lam, as follows: 1) an Ab responsewith isotype class switching, possibly induced by IL-4 secretion and2) a proinflammatory response through the activation of Th17 cellsinduced by the concomitant production of TGF-b and IL-6. Weconclude that our synthetic tripartite oligosaccharide conjugatevaccine equally promotes DC activation, the induction of the Th17response, and potent immunization.Several lines of evidence suggest that TS-TT-Lam will afford

protection. The isotype shift that results form DC targeting of thevaccine is important. It has been shown by several indirect anddirect measures that, in contrast to nonprotective mAbs, Abs thatprotect mice against C. albicans more efficiently bind complementfactor C3 to the yeast cell, and that the mechanism of protectionappears to be associated with enhanced phagocytosis and killingof the fungus (65). Our analysis of the IgG isotype distributionspecific for the b-mannan trisaccharide, although variable acrossthree strains of mice, shows a general shift toward IgG1 andsignificant levels of IgG2b with some IgG2a and IgG3. Literaturedata (66–68) indicate that IgG1 does not fix complement, butIgG2b does, as do the other two isotypes. Our own work (7)demonstrated that the glycopeptide of Ref. 10 (10), when con-jugated to tetanus toxin, yields anti-trisaccharide Abs with IgG2aand IgG3 subclass distribution and confers protection without adju-vant, presumably via the ability of the toxoid or its fragments tobe presented by DCs. The literature also reports that monocyteresponses to C. albicans are enhanced when C. albicans is opsonizedby specific Abs (69).Conjugate vaccines based on polysaccharides that combat

pneumococcal pneumonia, bacterial meningitis caused by Neis-seria meningitiditis, or Hemophilus influenza have been extremelysuccessful in reducing disease (70). Whereas these polysaccharidesconjugated to carrier protein are successful in raising protectiveAbs, smaller oligosaccharide epitopes are far less successful inconferring protection. Similar observations apply to tumor-associatedcarbohydrate Ags. A notable success in raising high titer of protectiveAbs has employed targeting of the innate immune system via TLRs.The approach we have adopted in this study does not require theactivation of TLRs and is consistent with reports of DC targeting ofprotein Ags.Avci et al. (64) have identified how glycoconjugate vaccines are

degraded after injection and then presented by cells of the immunesystem. These conclusions show that polysaccharide is degradedto ∼10-kDa fragments with attached peptide fragments, and thatthese fragments are recognized by TCRs. When conjugates werecreated that maximized the presentation of these essential ele-ments, a superior immune response was observed. This discoveryraises the prospect of designing fully synthetic three-componentconjugate vaccines composed of the optimum B cell epitope, aT cell peptide possibility replacing carrier protein with the thirdcomponent, a glycan able to direct the vaccine to DCs for effectivepresentation. A number of glycan receptors displayed on the DCmembrane are known to modulate immunological responses (71).Consequently, it might be expected that the most appropriate li-gand for targeting may vary depending on the particular vaccineand B cell epitope.Our combined approach of cellular immunology and in vivo

vaccination challenges confirms the importance of DCs in themodulation of immune responses. By carefully defining the path-ways stimulated by multicomponent vaccines, specific responses

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could be induced and regulated. Whereas further characterization ofthe T cells activated by these compounds is required, we will firstfocus on optimizing vaccine conjugates. In the context of a practicalC. albicans vaccine, we consider a trisaccharide B cell epitope (72)conjugated to the Fba 14 peptide (10) with attached multivalentb-glucan may provide an even more potent immunogenic com-pound.

DisclosuresD.R.B. is a founder and shareholder in a University of Alberta spinoff com-

pany, TheraCarb Inc., to which the vaccine described in Ref. 63 has been

licensed. The other authors have no financial conflicts of interest.

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