Journal of Controlled Release 172 (2013) 671–678

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Journal of Controlled Release

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β-Glucan microparticles are good candidates for mucosal antigendelivery in oral vaccination

Rebecca De Smet a,⁎, Tine Demoor b, Stephanie Verschuere a, Melissa Dullaers c, Gary R. Ostroff d,Georges Leclercq e, Liesbeth Allais a, Charles Pilette f, Marijke Dierendonck g,Bruno G. De Geest g, Claude A. Cuvelier a

a Department of Pathology, Ghent University, De Pintelaan 185, 9000 Ghent, Belgiumb Department of Pathology, University of Michigan, Zina Pitcher Place 109, 48109 Ann Arbor, USAc Laboratory of Immunoregulation and Mucosal Immunology, Department of Pneumology, Ghent University, De Pintelaan 185, 9000 Ghent, Belgiumd Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Suite 113, Biotech 2, Worcester, 01605, USAe Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, De Pintelaan 185, 9000 Ghent, Belgiumf Pole Pneumologie, ORL &Dermatologie, Institut de Recherche Expérimentale& Clinique and Department of Pneumology, Cliniques Universitaires St. Luc, Université Catholique de Louvain, AvenueHippocrate 54, 1200 Brussels, Belgiumg Laboratory of Pharmaceutical Technology, Department of Pharmaceutics, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium

Abbreviations: GP, β-glucan microparticles; PP, Peylymph nodes; S-IgA, secretory-immunoglobulin A; SC, secrecognition receptor; pIgR, polymeric immunoglobulin re⁎ Corresponding author at: Department of Pathology,

Pintelaan 185, 9000 Ghent, Belgium. Tel.: +32 9 332 36 8E-mail address: rebecca.desmet@ugent.be (R. De Sme

0168-3659/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.jconrel.2013.09.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 June 2013Accepted 5 September 2013Available online 14 September 2013

Keywords:β-glucan microparticlesAntigen deliveryOral vaccinationPeyer's patchesVaccines

Continuously improving the developmental process and the efficacy of oral vaccines is essential in the fight againstintestinal pathogens. A promising strategy for vaccination applying safe, biodegradable andnon-replicating antigendelivery systems has gained increased interest for eliciting cellular and humoral immune responses. The currentstudy evaluates the potential of β-glucan particles (GP) as an oral antigen delivery system and their adjuvant char-acteristics. GP are efficiently internalized by human intestinal epithelial cell lines (Caco-2 andHT-29 cells), withoutexerting negative effects on cell viability. GP triggered the expression of pro-inflammatory cytokines IL-23p19, IL-8and the β-glucan receptors dectin-1 and TLR2 by activated Caco-2 cells, and CCL20 in HT-29 cells. In contrast, theexpression level of TGF-β, an importantmediator of oral tolerance, was significantly downregulated in HT-29 cells.Additionally, adoptive transfer experiments showed proliferating ovalbumin (OVA)-specific CD4+T cellsmainly inthe spleens of GP-OVA-fed mice. Furthermore, we detected a significantly increased IL-17 and a trend towardsincreased IFN-γ production in the spleen of GP-OVA-fed mice upon antigen restimulation. Oral administration ofGP-OVA induced increasedOVA-specific IgA, secretory-IgA (S-IgA) and secretory component (SC) production in in-testinal fluids. Our data show that GP vehicles are able to deliver OVA via an oral route allowing efficient antigenpresentation alongside adaptive immune activation, resulting in a Th17-biased response and the production ofOVA-specific IgA, secretory-IgA and secretory component antibodies.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Parenteral vaccination strategies fail to generate an adequate localmucosal immune response against still prevailing intestinal infections,such as cholera, dysentery and typhoid fever. A topical–mucosal routeof vaccination is crucial to induce protective immune responses (mucosalS-IgA and serum IgG responses) and may even lead to immunity atdistant mucosal effector sites via the interconnected ‘common mucosalimmune system’ [1]. Particularly in developing countries, oral vaccina-tion may offer a means to deal with safety concerns (eliminates risk of

er's patches; MLN, mesentericretory component; PRR, patternceptor.5 Blok A, Ghent University, De6; fax: +32 9 332 49 65.t).

ghts reserved.

infections associated with needle re-use), poor patient compliance andthe need for mass vaccination.

However, the development of oral subunit vaccines holdsmajor chal-lengeswith regard to safety and efficacy in overcoming oral tolerance [2].First, the antigen delivery system must be designed to resist enzymaticdegradation by gastric and intestinal proteases and to deliver the antigenacross biological barriers — in particular the intestinal epithelium.Secondly, upon successful cellular uptake, the vaccine formulationneeds to elicit potent and long-lasting humoral and cellular immune re-sponses. Current oral vaccines are based on inactivated or attenuatedpathogens. Although both formulations provide a high level of antigenexposure and to some extent also have an intrinsic adjuvanticity, theirimmunogenicity and stability still remain a problem. In particular atten-uated vaccines maintain the risk of reversion to virulence and mayinduce disease in immunocompetent and immunocompromised indi-viduals. This could be counteracted by the use of recombinant/subunitvaccines based on specifically defined and purified antigens (peptides

672 R. De Smet et al. / Journal of Controlled Release 172 (2013) 671–678

or proteins), a safe alternative, however poorly immunogenic and oftenrequiring multiple doses and effective mucosal adjuvants [2].

The ideal vaccine carrier system needs to meet the desired require-ments for both the delivery and the adjuvant activity. Several nano-microparticulate systemswith intrinsic adjuvant and immunostimulatingactivity are currently evaluated as candidate carriers formucosal vaccinesin a vast range of animal models and human cell lines, including immunestimulating complexes (ISCOMs), liposomes, virosomes and polymericmicroparticles [3–5]. The potency and efficacy of these delivery systemsdiffer according to the animal model, organ system, in vitro/in vivo set-up, particle formulation and adjuvants used and many studies reportpromising outcomes. However, only poly(lactic-co-glycolic acid) (PLGA)nanoparticles and liposomes havemade it frompreclinical animal studiesto clinical trials so far [6].

Several other microparticles exhibit promising biological activity,but remain to be evaluated for use in the pharmaco-medical field.β-Glucan particles (GP), manufactured by the group of Ostroffet al., are purified from baker's yeast Saccharomyces cerevisiae andhave been shown tomodulate innate immunity and enhance tumoricidalactivity [7]. The molecular structure of β-glucans is recognized as apathogen-associated molecular pattern (PAMP) by pattern-recognitionreceptors (PRRs) such as dectin-1 and complement receptor 3 (CR3) onDCs, macrophages, monocytes and neutrophils [8,9]. Despite previousstudies implicating the adjuvant properties of β-glucans in antimicrobialand antitumor activity [10,11], their potential as vaccine carriers has notyet been thoroughly verified in human intestinal epithelial cell lines ororal immunization experiments. Versus other particulate delivery sys-tems, the GP delivery platform features efficient antigen loading (above90%) and receptor-targeteduptake intoAPCs combinedwith the inherentadjuvant functions of 1,3-β-D-glucans [12]. Beta-glucans are FDA ap-proved as generally recognized as safe (GRAS) [13]. GP allow efficienttrapping of the antigen retaining its conformation and integrity fortargeted antigen/adjuvant co-delivery to APCs. The GP size favors theiruptake byMcells in Peyer's patches (PP) and subsequently disseminationto the mesenteric lymph nodes (MLN), blood circulation and spleen [14].However, the current production procedure for GP vaccines resultsin liquid formulations, which maintains the need for cold-chain-constrained settings and therefore efforts are being made to developdry GP vaccine formulations.

The aims of this study were to first characterize the GP-antigenmicroparticles using ovalbumin (OVA) as a model antigen. Next weinvestigated the immunological interaction between GP-OVA andthe intestinal epithelium in in vitro patient-relevant epithelial celllines and in vivo oral immunization experiments. Subsequently, weinvestigated the cytokine and chemokine profiles produced inCaco-2 and HT-29 cell lines upon GP-OVA internalization, and the in-duction of MHCII mediated-antigen presentation using adoptive trans-fer experiments. Finally, an oral GP-OVA immunization experimentwas performed to assess whether humoral immune responses couldbe stimulated. Our results describe the efficacy and biocompatible fea-tures of amodel oral GP vaccine adding to their promise as a carrier sys-tem for oral vaccination of both animals and humans.

2. Material and methods

2.1. Synthesis of microparticles and protein encapsulation

Hollow β-glucan yeast shells (2–4 μm) were produced from com-mon baker's yeast, S. cerevisiae, by successive alkaline and acidic treat-ments as described by Soto and Ostroff [15]. Briefly, this procedurehydrolyzes the outer cell wall and intracellular components of theyeast cells, resulting in hollow yeast particles consisting of N85% β-1,6branched, β-1,3-D-glucans (β-glucans), 2% chitin and less than 1% lipidsand proteins [16]. BSA or OVA were encapsulated in GP (GP-BSA orGP-OVA) through lyophilization according to Huang et al. [17]. DryGP were hydrated with various BSA or OVA concentrations dissolved

in PBS for 2 h at 4 °C and subsequently frozen at −80 °C and lyophi-lized. This process is repeated and the dry-GP-BSA or dry-GP-OVA for-mulations were heated to 50 °C and swollen with 25 mg/ml tRNA(derived from torula yeast, type IV, Sigma Aldrich) in Tris buffer(pH 8) for 30 min and subsequently 10 mg/ml tRNA was added for1 h. Yeast tRNA forms a stable and insoluble complex that traps theBSA or OVA proteins inside the GP. The suspension was centrifugedand after three PBS washes, GP-BSA or GP-OVA were resuspended inPBS and stored at−20 °C.

2.2. Particle characterization and encapsulation efficiency

The surface and interior morphology of GP-OVA were examined byscanning electronmicroscopy (SEM)with a Quanta FEG FEI 200 appara-tus (FEI, Hillsboro, Oregon, USA) and by transmission electron micros-copy (TEM) with a Zeiss TEM900 microscope (Carl Zeiss, Oberkochen,Germany) at 50 kV. The surface charge of the GP-OVA was determinedby zeta-potentialmeasurementswith a Zetasizer nano series (Malvern).Particle size analysis was performed by laser diffraction using aMalvernMaster equipped with a RF300 objective. For OVA loaded beta-glucanmicroparticles, the unbound protein concentration in the supernatantwas measured using a Coomassie (Bradford) protein assay (ThermoFisher Scientific, Aalst, Belgium) according to the manufacturer's in-structions. The encapsulation efficiency is calculated as follows: Encap-sulation efficiency = 100 × (protein concentrationinitial − proteinconcentrationsupernatant / protein concentrationinitial). All measure-ments were conducted in triplicate.

2.3. In vitro experiments with Caco-2 and HT-29 cell cultures

The human colorectal adenocarcinoma cell lines Caco-2 and HT-29were purchased from the American Type Culture Collection (Rockville,MD, USA). Cell cultures were maintained in DMEM containing 10%FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamineand 0.1 mM non-essential amino acids, at 37 °C and 5% CO2. Caco-2cells were seeded on Thincert™ inserts (Greiner Bio-One, Wemmel,Belgium) at a cell density of 1 × 105 cells/well and cultured for 21 days.The integrity of the cellmonolayerswas verifiedby transepithelial electri-cal resistance (TEER) measurement with a Millicel-ERS Volt-Ohmmeter.Fully differentiated Caco-2 cells reached a TEER above 300–400 Ω cm2.HT-29 cells were seeded in a 24-well plate at a cell density of 3 × 105

cells/well and cultured for 5 days until a confluent monolayer wasachieved. A BSA-FITC loaded GP suspension (2.5 mg/ml and1.25 mg/ml) in DMEM culture medium was applied to the cellmonolayer and incubated for 3, 6 or 24 h.

2.4. Flow cytometry and confocal scanning laser microscopy

Following cell detachment by Accutase (eBioscience, Vienna, Austria)and FcR blocking with human FcR Blocking Reagent (Miltenyi Biotec,Bergisch Gladbach, Germany), single cell suspensions of Caco-2 andHT-29 cells were stained with CD324-APC (clone 67A4) (E-Cadherin)(Miltenyi Biotec, Bergisch Gladbach, Germany) and dead cells were ex-cluded by propidium iodide (0.1 mg/ml) (Invitrogen, Ghent, Belgium).CD86 expression was analyzed as a marker for activation of intestinalepithelial cells (IECs) using anti-CD86 (clone IT2.2) or isotype controlrat IgG2b, κ (both eBioscience, Vienna, Austria). Flow cytometric(FACS) evaluation of microparticle uptake and percentage of FITC fluo-rescent cells were performed on a LSR II cytometer equipped withFACSDiva software Version 6.1.2 (both BD Biosciences, Erembodegem,Belgium).

For confocal microscopy analysis, Caco-2 cells and HT-29 cells werecultured in a Lab Tek II eight-well glass chamber slide (Nunc, Roskilde,Denmark). Upon reaching confluence, the cell monolayers were incu-bated with BSA-FITC loaded beta-glucan microparticles for 24 h. Subse-quently, cells were washed with PBS and fixed in an aqueous 4%

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formaldehyde solution overnight, followed by nuclear staining with4′,6-diamidino-2-phenylindole (DAPI) and cell membrane staining withwheat germ agglutinin (WGA)-Texas Red®-X conjugate (20 μg/ml)(Invitrogen, Ghent, Belgium). Confocal images of three-dimensionalcultures were made with a Leica TCS SP5 AOBS confocal laser scanningmicroscope (CLSM).

2.5. In vitro cytotoxicity assays

The cytotoxicity of the β-glucan particles was evaluated using a col-orimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) assay. Briefly, Caco-2 cells and HT-29 cells were seeded ina flat-bottom 96-well plate (Greiner Bio-One) at 2 × 104 and 1 × 104

cells/well, respectively, in DMEM culture medium. Next, the cultureswere incubated with 1.25 and 2.5 mg/ml GP-OVA for 6, 12 or 24 h.The microparticles were removed by careful washing with PBS. Then,200 μl of MTT solution (0.5 mg/ml in PBS) was added and incubatedfor another 4 h at 37 °C. The reaction product was solubilized in200 μl DMSO and absorbance (Abs) was measured at 570 nm. DMEMculturemedium and 2% Triton-X100were used as negative and positivecontrols, respectively. The cell viability was calculated using the follow-ing formula: cell viability = (Abssample / Absnegative control) × 100. Theviability of untreated controls was normalized to 100%. Cytotoxici-ty = 100 − % cell viability. The particle/cell ratio was determined byBürker counting chamber calculations of the highest concentration(2.5 mg/ml) of the GP.

2.6. mRNA extraction, reverse transcription, and RT-PCR

mRNA was isolated from Caco-2 and HT-29 cell monolayers usingthe Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany), according tothe manufacturer's instructions. One microgram of RNA per samplewas reverse transcribed using the iScript cDNA synthesis kit (Bio-RadLaboratories, Hercules, CA, USA). Expression of target genes CD86,CD40, dectin-1, TLR2, IL-8, TGF-β, CCL20 and IL-23p19 was analyzedwith the Sensimix SYBRNo-ROX kit (Bioline, London, UK). The followingforward and reverse primers were used: 5′-AAACTCGCATCTACTGGCAAA-3′ and 3′-GGTTCTTGTACTCGGGCCATA-5′ (human CD80); 5′-ACTGAAACGGAATGCCTTCCT-3′ and 3′-CCTCACTCGTACAGTGCCA-5′ (humanCD40); 5′-TCAATGTAAGAGGAAGGGTG-3′ and 3′-GCCAAGCTCTCTAAACATTT-5′ (human dectin-1); 5′-TGCGGAAGATAATGAACACC-3′ and 3′-GATCCCAACTAGACAAAGACTG-5′ (human TLR2); 5′-TGTTCCACTGTGCCTTGGTTTC-3′ and 3′-TGTGAGGTAAGATGGTGGCTAATAC-5′ (human IL-8); 5′-GGCCAGATCCTGTCCAAGC-3′ and 3′-GTGGGTTTCCACCATTAGCAC-5′ (human TGF-β); 5′-TGCTGTACCAAGAGTTTGCTC-3′ and 3′-CGCACACAGACAACTTTTTCTTT-5′ (human CCL20) and 5′-TAGCCGCCCGGGTCTT-3′ and 3′-TGGAGATCTGAGTGCCATCCT-5′ (human IL-23p19). Geneexpression was normalized to two reference genes (HPRT, RPL13A).

2.7. Animals

Male OT-II mice (6–9 weeks old), carrying a transgenic CD4 T cellreceptor (TCR) specific for the MHCII-restricted OVA peptide aa 323–339 [18], were kindly provided by Dr. Johan Grooten (Ghent University,Ghent, Belgium). Male C57BL/6 wild-type (WT) mice (6–9 weeks old)were obtained from Charles River Laboratories France. The local EthicsCommittee for animal experimentation of the faculty of Medicine andHealth Sciences (Ghent University, Ghent, Belgium) approved allin vivo manipulations.

2.8. Ligated ileal loops

OVA-Alexa Fluor 488 (AF488) loadedGP (200 μl)were administeredto male wild type C57BL/6 mice (5 animals per group) via ligated ilealloops, enclosing PP, at a particle concentration of 1 × 108/ml. Onehour later, transepithelial particle transport and uptake in the PP were

evaluated by CLSM and TEM. For CLSM analysis, cryosections of PP wereimmunofluorescently stained using TRITC-conjugated Ulex europaeusagglutinin 1 (UEA-1) (Sigma-Aldrich) as a murine M-cell marker andDAPI nuclear staining, as described previously [19]. Samples of PPs wereprocessed for TEM analysis as described by Verschuere et al. [20] and im-agedwith a Zeiss TEM900microscope (Carl Zeiss, Oberkochen, Germany)at 50 kV.

2.9. In vivo CD4+ T cell activation

CD4+ T cells from spleens of OT-II mice were negatively enriched bymagnetic cell sorting (MACS) using the CD4+ T cell isolation kit (MiltenyiBiotec, Bergisch Gladbach, Germany). CD4+ T cells were resuspended inPBS containing 0.1% BSA (SigmaAldrich) at 1 × 107 cells/ml andwere in-cubated in a final concentration of 5 μMcarboxyfluorescein succinimidylester (CFSE) for 15 min at 37 °C. Further uptake of the dye was blockedby the addition of cold PBS containing 30% FCS. The cells were washedthree times, with the last wash being performed in serum-free PBS.Finally 200 μl of cells resuspended at a density of 5 × 106 in PBSwas i.v. injected in recipient WT C57BL/6 mice. After 24 h, the micewere orally immunizedwith either PBS, 10 mg soluble OVA or encapsu-lated in GP (GP-OVA 10 mg). Three days later, mice were euthanizedand single cell suspensions of PP, MLN and spleen were analyzed byFACS to determine the percentage of proliferated CD3+ CD4+ T cells de-fined by CFSE fluorescence intensity. 2 × 105 PP, MLN and spleen cellswere seeded in 96-well plates and restimulated with 100 μg OVA/mlfor 72 h. Secreted cytokine levels were determined in culture superna-tant using a Bio-Plex assay (Bio-Rad).

2.10. Oral immunization model

C57BL/6 WT mice received oral doses of either 200 μl PBS, solubleOVA (100 μg) or GP-OVA (low dose or high dose GP-OVA comprising100 μg or 300 μg OVA respectively) for three consecutive days. Boosterdoses were given on days 14 and 21. Before each immunization, foodbut not water was withheld overnight [21–23]. On day 28, blood andintestinal lavage fluid were collected as described previously [24,25],for the analysis of IgG and IgA titers in sera and intestinal lavage super-natants as described below.

2.11. Determination of total and OVA-specific IgG and IgA by ELISA

A commercially available ELISA kit was used to determine OVA-specific IgG antibody titers in serum (Alpha Diagnostic International,San Antonio, TX, USA) and results are expressed as U/ml. OVA-specificIgA antibodies were determined in serum and intestinal lavage super-natant using an in-house ELISA assay. OVA (Sigma Aldrich) was coatedat 100 μg/ml and following a blocking stepwith 1% BSA in PBS, serial di-lutions of serum and intestinal lavage samples were added in duplicate.Goat anti-mouse IgA-HRP (Southern Biotech, 1/5000 dilution)was usedfor detection. Following washing, the substrate solution containinghydrogen peroxide/ultra 3,3′,5,5′-tetramethylbenzidine (TMB) (Pierce,IL, USA) was added to each well for 30 min and the reaction wasstopped with 2.5 N H2SO4. The optical density (OD) was measured at450 nm and data are expressed as corrected OD (absolute OD givenby the sample corrected for the background), in the absence of amurinestandard. Secretory component (SC) was assessed using a direct ELISAusing goat anti-mouse pIgR (R&D Systems), as capture antibody andHRP-conjugated anti-goat IgG (Sigma Aldrich) for detection. Total andSecretory IgA (S-IgA) were measured in intestinal lavages with a sand-wich ELISA. Samples were assayed using a polyclonal goat anti-mousepIgR or anti-mouse IgA (Sigma Aldrich), as capture antibody andbiotinylated goat anti-mouse IgA (Sigma-Aldrich) as detection antibody[26,27]. Results of intestinal total IgA and SC and were expressed asμg/ml and ng/ml, respectively. Intestinal S-IgA data are expressedas corrected OD. The optical density cut-off point was 0.16 for serum

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OVA-specific IgA, 0.3 for intestinal OVA-specific IgA and 0.07 for intesti-nal S-IgA. For serumOVA-specific IgG, intestinal total IgA and intestinalSC, a cut-off value of respectively 40 U/ml, 60 ng/ml and 100 pg/mlwasused.

2.12. Statistical analysis

Reported values are expressed as mean ± SEM. Statistical analysiswas performed by SPSS 20 Software (SPSS 20 Inc., Chicago, IL, USA)using non-parametric tests (Mann–Whitney U). For comparison ofmore than two groups, use was made of one-way analysis of variance(ANOVA), followed by post hoc Bonferroni tests or non-parametricKruskal–Wallis tests if conditions for ANOVA were not met. A P-valueb0.05 was considered significant.

3. Results

3.1. Microparticle characterization and encapsulation efficiency

Fig. 1 illustrates the surface and interior morphology of GP-OVA,which were visualized by scanning (Fig. 1A) and transmission electronmicroscopy (Fig. 1B).

GP-OVA can be distinguished by their characteristic ellipsoidal“wrinkled pea” morphology and irregular delineation, demonstratingthe hydrolysis of the outer cell wall and intracellular components attrib-uted to the consecutive alkali and acid treatments. On TEM, the encapsu-lated OVA/yeast tRNA complexmanifests itself as a dark gray granularityin the core of the GP further demonstrating successful OVA encapsula-tion inside the GP. The size of the GP-OVA was determined by laser dif-fraction measurements revealing a mean diameter of 3.7 ± 0.2 μm(Fig. 1C) and GP-OVA were negatively charged as indicated by thezeta-potential (−6.4 ± 0.3 mV). The GP delivery platform features effi-cient antigen loading and an encapsulation efficiency up to 97.8 ±2.0%was achieved using a GP/OVA ratio of 2/1 (Fig. 1D).

3.2. In vitro microparticle uptake by human intestinal epithelial cells

A prerequisite for an oral vaccine delivery system is to be efficientlyinternalized by the intestinal epithelium and therefore we investigated

Fig. 1. Microparticle characterization and encapsulation efficiency of OVA loaded beta-glucan microparticles. (A) SEM image, (B) TEM micrograph (the insets show GP-OVA ata higher magnification) and (C) size distribution of GP-OVA. (D) Encapsulation efficiencyof the GP-OVA/yeast tRNA formulation for different amounts of OVA added to 1 mg ofbeta-glucan microparticles.

the interaction between the β-glucan-based carrier system and differenthuman epithelial cell lines. Caco-2 cellsmodel the epitheliumof the smallintestine, whereas HT-29 cells resemble the epithelium of the large intes-tine, and both cell lines are commonly used for in vitro modeling of per-meability, transport and uptake of components upon pharmaceuticalintervention [28]. Caco-2 and HT-29 cells were incubated with BSA-FITCloaded GP at particle concentrations of 1.25 mg/ml and 2.5 mg/ml(optimal concentrations determined in MTT assay) for 3, 6 or 24 h[29]. The efficiency of particle uptake was assessed using flow cytome-try, via particle modification with FITC-conjugated BSA and cell stainingfor the epithelial marker CD324. The beta-glucan microparticles weretaken up efficiently by both IEC lines, with HT-29 cells being more effi-cient in general compared to Caco-2 cells (Fig. 2A). After 24 h of incuba-tion, the uptake of GP was 1.38-fold and 1.59-fold higher, at 1.25 mg/mland 2.5 mg/ml respectively, in HT-29 cells compared to Caco-2 cells(both P b 0.001). Cellular uptake of the BSA-FITC loaded GP increasedwith incubation time and was dose-dependent (1.25 and 2.5 mg/ml).

Subsequently, confocal microscopy was used to confirm the flowcytometric results in these two cell lines after 24 h of incubation withBSA-FITC loaded GP. This allowed us to distinguish between internaliza-tion of the particles and extracellular binding to the cell surface, and todetermine the exact localization of particles within the cell. As shown inFig. 2B and C, the BSA-FITC loaded GP, identified as green fluorescentspots, are within the cell boundaries at different depths from the apicalsurface and the majority of the internalized microparticles were clus-tered in the apical and perinuclear regions of the cells. These datashow that GP-based antigen delivery vehicles are efficiently internal-ized by IECs and mainly localized in the apical and perinuclear cellcompartments.

3.3. The effect of particle loading on human intestinal epithelial cell viability

To examine a possible cytotoxic effect of GP upon uptake by the in-testinal epithelium, we performed a standardMTT viability assay. Mito-chondrial dehydrogenase activity (MDA) of the Caco-2 cells and HT-29

Fig. 2. In vitromicroparticle uptake by human intestinal epithelial cells. (A) BSA-FITC loadedGP were incubated with Caco-2 and HT-29 cells at particle concentrations of 1.25 and2.5 mg/ml for 3, 6 or 24 h. Representativedata of two independent experiments in triplicateare shown (expressed as percentage FITC-positive cells, mean ± SEM). *, P b 0.05; **,P b 0.01; ***, P b 0.001. Confocal photomicrographs of Caco-2 cells (B) and HT-29 cells(C) following a 24 h incubationwith BSA-FITC loadedGP (full arrows). BSA-FITC loadedpar-ticles (green); plasma-membrane marker wheat germ agglutinin (WGA)-Texas Red®-X(red); DAPI staining (blue).

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cells was evaluated as ameasure for cytotoxicity at different time points(6, 12 and 24 h) after incubation with 2.5 or 1.25 mg/ml of GP-OVA(Fig. 3A). GP-OVA did not affect the viability of the Caco-2 cells(99.6%), even at the highest investigated concentration (2.5 mg/ml). Asignificant increase of the MDA at highest GP-OVA concentration at24 h of incubation was observed in Caco-2 cells, compared to untreatedcontrols (P b 0.001). Especially in the HT-29 cell line, an activation ofthe MDA was observed at 6 h incubation at 2.5 mg/ml, however, ex-tended incubation periods resulted in a decreased cell viability (70.9%).

3.4. GP induce the expression of chemokines, pattern recognition receptorsand costimulatory molecules in human intestinal epithelial cells

Intestinal epithelial cells are crucial players in the initiation ofmuco-sal immune responses. They are thefirst line of defense against invadingpathogens for which they are endowed with PRR. Subsequently theyproduce an array of chemokines and cytokines to attract and activateimmune cells [30]. We investigated the effect of interactions betweenepithelial cells and GP-OVA on the chemokine and cytokine profiles byRT-PCR (Fig. 3B–F). Caco-2 cells and HT-29 cells were cultured for 6 hat a particle concentration of either 1.25 mg/ml or 2.5 mg/ml GP-OVA.Subsequently, we chose to investigate themRNA and protein expressionof molecules characteristic for antigen presentation (HLA-DR, CD40,CD86), the main beta-glucan receptors (dectin-1, CR3 and TLR2), oraltolerance (TGF-β) and molecules steering the differentiation of Th17cells (IL-23p19) and chemotaxis of innate immune cells and APCs (IL-8and CCL20).

Fig. 3. GP-OVA induced expression of chemokines, pattern recognition receptors andcostimulatory molecules in human intestinal epithelial cells. (A) The biocompatibility ofGP-OVA with Caco-2 and HT-29 cells was monitored by measuring the mitochondrial de-hydrogenase activity at particle concentrations of 1.25 mg/ml and 2.5 mg/ml, at differenttime points of in vitro culture (6, 12 and 24 h). Results are expressed as percentage of mi-tochondrial activity, taking as 100% the mitochondrial activity of untreated controls at allthree time points. Representative data of three independent experiments in triplicateare shown. (B) mRNA levels of CCL20 and TGF-β in HT-29 cells upon culture for 6 hwith 1.25 mg/ml and 2.5 mg/ml GP-OVA. (C) GP-OVA induced mRNA expression ofdectin-1 and TRL2 in Caco-2 cells. mRNA and protein expression of (D) CD40 and (E)CD86, (F) mRNA expression of IL-8 and IL-23p19 in Caco-2 cells cultured for 6 h at a par-ticle concentration of either 1.25 mg/ml or 2.5 mg/ml. mRNA expression is indicated rela-tive to the expression of two reference genes HPRT and RPL13A (mean ±SEM, n = 2).Flow-cytometric histograms of CD40 and CD86 protein expression are representative of4 replicates per group. *, P b 0.05; ***, P b 0.001. NC = negative control.

mRNA expression of CCL20 and TGF-β was significantly altered inHT-29 cells (Fig. 3B), whereas these changes were not observed inCaco-2 cells (data not shown). mRNA levels of CCL20/macrophage in-flammatory protein (MIP)-3α, an attractant for immature DCs and lym-phocytes, was significantly increased in HT-29 cells exposed to GP-OVA(P b 0.001, negative control (NC) and GP-OVA 1.25 mg/ml 6 h)(P b 0.001, NC and GP-OVA 2.5 mg/ml 6 h). Interestingly, the expres-sion level of TGF-β, an important mediator of oral tolerance, was signif-icantly downregulated (P = 0.014) in HT-29 cells following GP-OVAincubation.

The expression of receptors involved in GP-OVA recognition, dectin-1 and TLR2, were both highly increased in Caco-2 cells (Fig. 3C). Thesereceptors, respectively belong to the family of C-type lectin (CRLs) andToll-like receptors (TLRs) and collaborate in the production of inflam-matory cytokines and chemokines [31,32]. Another main membranebeta-glucan receptor termed CR3 was not significantly increased uponGP-OVA incubation (data not shown). Although GP-OVA did not induceincreased HLA-DR expression (data not shown), the activation statusof Caco-2 cells was enhanced especially at the highest GP-OVA con-centration as shown by significantly increased mRNA expression levelsof the co-stimulatory molecules CD40 (P b 0.001) and CD86(P b 0.001) compared to untreated controls. Increased CD40 and CD86expression were confirmed at the protein level (Fig. 3D and E).Interleukin-23 p19 subunit (IL-23p19) enhances the expansion of earlyTh17 committed cells and their terminal differentiation into IL-17 effec-tor cells [33]. IL-23p19 and IL-8mRNA levelswere upregulated in Caco-2cells, demonstrating a Th17 biased response and attraction of innate im-mune cells upon antigen internalization, respectively (Fig. 3F). Together,these data show that GP-OVA did not affect the viability of IECs and in-duced increased mRNA expression levels of molecules engaged inbeta-glucan recognition, antigen presentation and differentiation to-wards Th17 biased responses in activated human IECs.

3.5. In vivo transepithelial uptake of OVA-AF488 loaded GP occurs byM cells in the Peyer's patches (PP) of the murine small intestine

In order to determinewhether transmucosal uptake of antigen loadedGP occurred in mice, we performed intestinal ligated loop experimentsand checked subsequent antigen presence in the PP. M cells, atypical ep-ithelial cells specialized in antigen transcytosis through the epithelial bar-rier into the PP, were visualized by U. europaeus agglutinin 1 (UEA-1), a murine M cell marker (Fig. 4A and B). After 1 h of incubation,CLSM demonstrated that the OVA-AF488 loaded GP were localizedeither intraepithelially, in the subepithelial dome (SED) or intraluminally,however itwas inconclusivewhether particle transport occurred trans- orparacellular and via M cell-mediated transcytosis. Therefore, TEM wasperformed and micrographs (Fig. 4C–E) showed the endocytosis ofGP-OVA through the transcellular pathway in M cells within thefollicle-associated epithelium (FAE). M cells play a key role in continu-ous sampling of antigens from the lumen and transporting the antigentowardsmucosal lymphoid tissues [34]. The results of the in vivo ligatedloop experiments demonstrated that OVA-AF488 loaded GP uptake oc-curred through the transcellular pathway in M cells.

3.6. OVA delivery via GP induces in vivo proliferation of OVA-specificCD4+ T cells

To investigate whether the uptake of microparticulate GP-OVA canstimulate MHCII mediated-OVA presentation, we analyzed in vivo(OT-II) proliferation of adoptively transferred CFSE-labeled OVA-specif-ic T cells. As illustrated in Fig. 5A, 5 × 106 CFSE-labeled CD4+ T-cells(purified from the spleen of OT-II mice) were injected i.v. into wild-type C57BL/6 mice. 24 h after the T cell transfer, the mice receivedPBS, soluble OVA or orally administered GP-OVA. Soluble OVA wastaken along as control. After three days, PP, MLN and spleens wereharvested and analyzed by flow cytometry. By means of CFSE labeling

Fig. 4. Transepithelial uptake of OVA-AF488 loaded GP by M cells in murine Peyer'spatches. (A and B) Representative confocal photomicrographs illustrating the presenceof OVA-AF488 loaded GP in the lumen of the small intestine (L), in the follicle-associatedepithelium (FAE) and in the subepithelial dome (SED) of a Peyer's patch (PP), 1 h after in-jection of GP into ligated ileal loops (200 μl at a particle concentration of 1 × 108/ml).OVA-AF488 loaded beta-glucan particles (green); M cell markerUlex europaeus agglutinin1 (UEA-1) (red); DAPI staining (blue). For the same experimental conditions, TEM photo-micrographs demonstrate the transcytosis of GP-OVA through M cells of the FAE (C–E).Lymphocytes (L) are also present in the intraepithelial pocket of the M cell. N = nucleus.

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of OT-II cells, we were able tomonitor OT-II cell migration and to deter-mine the frequency of adoptively transferred cells that had entereddivision, because of the sequential decrease in fluorescent CFSE labelingin daughter cells. Fig. 5B is a quantitative representation of OT-II celldivision and shows that three days after feeding, GP-OVA induced mi-gration and proliferation of OVA-specific T cells in PP, MLN and spleenas compared to the PBS group. However, more divided OVA-specificcells (51.5 ± 11.2%) were found in the spleen (P = 0.009) of GP-OVA-fed mice in comparison with the PBS group, whereas solubleOVA-fed mice mainly showed dividing OT-II cells in PP (47.6 ±10.3%) and MLN (46.5 ± 10.6%). In order to determine the type ofT helper response induced by the GP antigen delivery, we measuredthe secreted cytokine levels characteristic for Th1, Th2 and Th17using a Bio-plex assay, after in vitro OVA restimulation of PP, MLN

Fig. 5.GP-OVA induces high OT-II cell proliferation and IL-17 expression in vivo. (A) Adop-tive transfer protocol. (B) The percentage of divided CFSE-labeled CD4+ T cells in thePeyer's patches, mesenteric lymph nodes and spleen is indicated (mean ± SEM, n = 5).(C) IL-17 and (D) IFN-γ secretion upon re-stimulation of splenocytes with OVA (100 μg/ml) for 72 h, determined by a Bio-Plex assay (expressed as pg/ml; mean ± SEM). *,P b 0.05; **, P b 0.01, ***, P b 0.001.

and spleen cells for 72 h. Interestingly, in line with the increased OT-IIproliferation, we observed a significantly stronger increase of the IL-17production in the splenocytes of GP-OVA-fedmice, as compared to solu-ble OVA-fed mice (P = 0.016), whereas IL-17 produced by PP and MLNof GP-OVA-fed mice were only just above the detection limit (Fig. 5C).IFN-γ (Fig. 5D) follows the same trend, although not significant. Slightincreases of both IL-17 and IFN-γ cytokines are observed in the PP andMLN of soluble OVA-fed mice, however not significantly different incomparison with GP-OVA-treated mice. These results demonstrate thatoral GP-OVA induces OT-II proliferation in the spleen and is accompa-nied by an increased IL-17 production and a tendency towards increasedlevels of IFN-γ, characteristic for a mixed Th17/Th1 immune response.

3.7. Increased intestinal antigen-specific IgA titers and transepithelialtransport of S-IgA upon oral immunization with GP-OVA

Because of the favorable in vitro characteristics of theGP-OVAdeliveryplatform, in particular their 3–4 micron size, efficient antigen encapsula-tion, highly efficient internalization by the intestinal epithelium andMHCII-mediated antigen presentation in vivo, we used GP-OVA in anoral immunization experiment. The goals were to determine if oralGP-OVA immunization leads to the delivery of the encapsulated antigen(OVA) and induces local and systemical humoral immune responses, inparticular antigen-specific antibodies. As illustrated in Fig. 6A, differentgroups of mice received either PBS, soluble OVA and low dose or highdose GP-OVA by oral gavage. Sensitization doses were given for threeconsecutive days and booster doses on days 14 and 21. Mice wereeuthanized on day 28 and total and OVA-specific IgG and IgA titers insera and intestinal lavage fluids were analyzed by ELISA. OVA-specificIgG and IgA levels in serum and intestinal total IgA levels were notincreased in GP-OVA-treated mice in either the low or high doseGP-OVA treated group (Fig. 6B–D). However, antigen-specific anti-bodies in the intestinal lavage were raised in the high dose GP-OVA-treated group (P = 0.027) compared to PBS group (Fig. 6E). Secretory

Fig. 6. Systemic and mucosal antibody responses upon oral GP-OVA immunization. (A)Oral immunization protocol. (B) OVA-specific IgG (C) OVA-specific IgA levels in serum;(D) total IgA; (E) OVA-specific IgA; (F) secretory IgA (S-IgA) and (G) secretory component(SC) levels in IL of PBS ( ), OVA ( ), low dose GP-OVA ( ), high dose GP-OVA ( ) treat-ed animals, as measured by ELISA. OVA-specific IgA in IL and serum and S-IgA levels in ILare expressed as corrected optical density (OD)(mean ± SEM, n = 8) and OVA-specificIgG in serum, total IgA and SC levels respectively as U/ml, μg/ml and ng/ml. *, P b 0.05;**, P b 0.01 compared to the PBS-fed group; ##, P b 0.01 compared to the OVA-fedgroup. IL = intestinal lavage.

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component (SC) is a glycoprotein resulting from pIgR transcytosis, en-hancing the immune/scavenging functions of S-IgA, and both are sug-gested to serve as competitive inhibitors of pathogen binding to hostcells [35,36]. Similarly with the intestinal OVA-specific IgA levels,the S-IgA production was significantly increased in the high doseGP-OVA-fed animals (P = 0.024) (Fig. 6F). Interestingly, a significantincrease was observed in intestinal lavage SC levels of the low doseand high dose GP-OVA group (respectively P = 0.011 and P = 0.005,compared to the PBS group and P = 0.007 and P = 0.003, comparedto the OVA-fed group) (Fig. 6G). We conclude that only multiple oraladministrations with the high dose GP-OVA caused an increase of theintestinal OVA-specific IgA and secretory-IgA levels in mice. However,as for both low dose and high dose GP-OVA-fed groups significantly in-creased levels of the secretory component are seen in comparison withPBS and soluble OVA-fed mice.

4. Discussion

In this study,we demonstrated highly efficient internalization of beta-glucan microparticulate antigen carriers by the intestinal epithelial–mucosal interface, both in vitro and in vivo. The beta-glucan particle(GP) uptake was paralleled by an increase in the receptors dectin-1 andTLR2 and resulted in increased expression of proinflammatory cytokinesand chemokines. Moreover, GP exerted adjuvant properties, as reflectedby a specific humoral S-IgA production and promoted Th17 responsein vivo.

It is well-known that the pharmaceutical world is copingwithmanyhurdles in the development of oral vaccines, especially with regard tothe harsh gut environment and oral tolerance. Biodegradable particu-late delivery systems are more effective than soluble antigens in stimu-lating protective immunity, and thus are a promising vaccinationstrategy. However several issues need to be addressed during theirdevelopment.

A first concern is whether the intestinal epithelium is able to inter-nalize the particles.We confirmed the efficient and rapid internalizationoffluorescent beta-glucan basedmicroparticle vaccines by two differenthuman intestinal epithelial Caco-2 and HT-29 cell lines in vitro usingFACS and CLSM analyses as previously modeled by the in vitro Caco-2/Raji M-cell model system [37]. GP vaccines are of an ideal size of naturalfungal pathogens (3–4 micron size and surface characteristics) to berecognized and internalized by M-cells in Peyer's patches [38] andtheir beta 1,3-D-glucan surface composition is recognized by dectin-1and TLR2 providing for receptor-targeted antigen delivery to dendriticcells in gut associated lymphatic tissues [39]. In addition to our in vitroexperiments, in vivo ligated intestinal loops confirmedOVA-AF488 loadedGP internalization that was M cell-mediated and occurred throughtranscellular pathways, as demonstrated by TEM. This is in agreementwith a study by Awaad et al. [40] that describes uptake of thiol-organosilica particles by the transcellular M-pathway in PP.

A second point of interest in vaccine design is to assure that the in-teraction of antigen delivery vehicles with the intestinal epitheliumdoes not exert cytotoxicity. Toxicity was non-existent in the Caco-2cell line at effective particle concentrations and minimal after extendedincubation periods with HT-29 cells. Nonetheless, we believe that theseconcentrations will never be achieved for such a long period underphysiological conditions, given the dilution, enzymatic degradation,muco-adhesion and the intestinal transit in vivo. Indeed, the mice in-cluded in the oral gavage experiment, tolerated the GP-OVA formula-tion well after repeated oral administrations. In general, the oraladministration of GP-OVA can be considered as safe.

Once the GP-OVA are internalized by intestinal epithelial cells, thequestion ariseswhich immunepathways are triggered andhow themu-cosal immune system responds. It is desirable that particulate antigencarriers trigger PRRs, hence activating the innate and adaptive immunesystem. After GP-OVA administration with the highest concentration(2.5 mg/ml), a marked induction of the beta-glucan receptors, dectin-1

and TLR2, and chemokine IL-8 were observed, accompanied by increasesin IL23-p19 and costimulatory molecules and importantly a decrease inTGF-β expression levels. As for the chemokine CCL20, both 1.25 and2.5 mg/ml GP-OVA concentrations were able to induce an increasedgene expression. We hypothesize that upon GP-OVA recognition,TLR/CLR triggering at the level of IECs and M cells in Peyer's patchesis one of the earliest events and induces the release of chemokines andpro-inflammatory cytokines, attracting innate immune cells and skewingtowards a Th17 immune response.

Although GP-OVA did not induce increased HLA-DR expression(data not shown), they clearly enhanced the activation status of theIECs. Indeed, according to several groups, IECs use HLA Class II pathwaysfor antigen processing [41,42]. The latter is suggestive for a role of IECsin sampling luminal GP and upon processing and presentation, in stim-ulating or augmenting T cell responses. Concomitantly, overcoming oraltolerance is amajor barrier to the development of effective oral vaccines.The marked reduction of TGF-β, as evidenced by RT-PCR, may impedethe ability of DCs to produce suppressive cytokines and the inductionof Treg cells in the MLN, thereby fostering the induction of an effectiveimmune response to the orally delivered antigen.

We believe that a pivotal role is reserved for IL-17-secreting Th17cells, in promoting mucosal IgA responses upon oral GP-OVA adminis-tration. Importantly, ex vivomeasurement of cytokine profiles revealedhigh IL-17 levels and a tendency towards increased levels of IFN-γ insplenocytes of GP-OVA-fed mice, but not in their PP and MLN cells,resulting in a tendency towards a mixed Th17/Th1 immune response.This suggests that, upon GP presentation by PP IECs and M cells andtogether with IL23-p19 production, naïve CD4 T cells differentiated toIL-17-secreting Th17 cells. The latter may have entered the bloodcirculation, preferentially migrating to the spleen and subsequentlyhoming towards the gut germinal B centers inducing the classswitch of B cells towards IgA-production. Cells that underwentmore than 3 divisions could not be detected in our assay setup, soit is well possible that these are present in PP of GP-OVA mice.This is in line with a previous study by Cao et al. [43] describingthat intestinal IgA secretion in TCR-βxδ−/− mice was promotedupon repletion of Th17 cells and by Huang et al. [17] who recentlydescribed Th1–Th17 biased cellular responses following subcutaneousGP vaccine administration.

A last important issue in the development of antigen carriers, iswhether antigen carriers are able to induce potentmucosal adaptive re-sponses. Interestingly, adoptive transfer experiments indicated morevigorous proliferation of OVA-specific CD4+ T cells in the spleen ofGP-OVA-fed mice and to a lesser extent in their PP and MLN andvice versa in the case of soluble OVA-fed mice. We hypothesize thatcarrier-derived Ag is primarily presented in the PP and MLN and ac-tivates CD4+ T cells, which subsequently circulate systemically andmigrate to distant sites, such as the spleen. In addition, we suggestthat GP particulate antigen is more efficiently internalized andpresented in a class-II dependent pathway. Themost plausible explana-tion for the more efficient Ag presentation in OVA-specific CD4+ T cellsis that it results from a more efficient glucan receptor-mediated uptakeinto antigen presenting cells, combined with a controlled release of Agfromparticulate GP comparedwith soluble Ag. Ourmost striking findingfrom the oral gavage experiment is the significantly higher intestinalS-IgA in the high dose GP-OVA-fed group and SC levels in both lowand high dose GP-OVA-fed animals. Thismay be indicative of a higherproduction by IgA-committed B-cells and higher local active transportof IgA across the epithelium related to enhanced expression of epithelialpIgR. In addition, OVA-specific antibodies were increased in the intesti-nal fluid of high dose GP-OVA-fedmice.We assume that the low dose ofGP was insufficient to pick up OVA-specific systemic antibodies. As em-phasized by our study, we believe that GP administration triggered PRRsignaling and consequently specific chemokine and cytokine profilesaccompanied with Th17-polarized responses, probably exerting crucialroles in inducing the remarked mucosal IgA responses. Future research

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needs to elucidate the underlyingmechanisms of this Th17-IgA axis andfocus of the use of clinical relevant antigens in the context of pathogenchallenge of microparticle immunized mice.

In conclusion, this study highlights the competence of beta-glucanmicroparticles to serve as an oral antigen delivery system. We showthat intimate contact between the antigen carrier systemand themuco-sal interface results in the production of pro-inflammatory cytokinesand chemokines. Subsequently, proliferating antigen-specific CD4+ Tcells enter the systemic circulation and spleen in GP-OVA-fed miceand promote Th17-polarized responses. Oral immunization withGP-OVA leads to robust humoral responses in the small intestinecomprising of high S-IgA and SC production/transport. Our findingsregarding a GP-based vaccine strategymight be crucial in the currentdevelopment of oral vaccination against enteric pathogens.

Acknowledgments

This work was supported by the Concerted Research Action of theUniversity of Ghent (BOF10/GOA/21, project number: 01GC2110W;Ghent, Belgium). We are grateful to Eef Parthoens for providing helpwith the confocal laser scanning microscope. Male OT-II (C57BL/6)TCR transgenic mice were kindly provided by Muriel Smet and Prof.Dr. Johan Grooten. We thank Dorothea Van Limbergen and Ran Rumesfor the processing of the samples for transmission electron microscopy,Sophie Vermaut for the helpwithflow cytometry experiments and LynnSupply, Marie-Rose Mouton and Gabrielle Holtappels for the excellenttechnical support. We are grateful to Prof. Jean Plum, Magda De Smedtand Stefaan De Koker for their critical revision and helpful discussions.

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