Bioconjugate Chem. 2006, 17, 1136−1140
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Synthesis and in Vitro Characterization of Antigen-Conjugated Polysaccharide as a CpG DNA Carrier Naohiko Shimada,† Ken J. Ishii,§,⊥ Yoichi Takeda,† Cevayir Coban,§,| Yuichi Torii,⊥ Seiji Shinkai,‡,# Shizuo Akira,§,⊥,| and Kazuo Sakurai*,† Department of Chemical Processes & Environments, The University of Kitakyushu, 1-1, Hibikino, Wakamatu-ku, Kitakyushu, Fukuoka 808-0135, Japan, Department of Chemistry & Biochemistry, Graduate School of Engineering, and Center for Future Chemistry, Kyushu University, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan, and Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency (JST), and Department of Host Defense and The 21st Century COE, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan. Received March 20, 2006; Revised Manuscript Received July 4, 2006
Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) are known as an immune adjuvant. CpG DNAs coupled with a particular antigen enabling both CpG DNA and antigen delivery to the same antigenpresenting cell have been shown to be more effective. Based on our previous finding that β-(1f3)-D-glucan schizophyllan (SPG) can be used as a CpG DNA carrier, here we present the synthesis of an antigen-conjugated SPG and the characterization of the conjugate. Ovalbumin (OVA, 43 kDa) was used as a model antigen, and two OVA were conjugated to one SPG molecule (Mw ) 150 000), denoted by OVA-SPG. Circular dichroism and gel electrophoresis showed that OVA-SPG could form a complex with a (dA)40-tailed CpG DNA at the 3′ end (1668-(dA)40). When OVA-SPG was added to macrophages (J774.A1), the amount of the ingested OVA-SPG was increased compared with that of OVA itself, suggesting that Dectin-1 (proinflammatory nonopsonic receptor for β-glucans) is involved to ingest OVA-SPG. Furthermore, the complex of the conjugate and DNA was colocalized in the same vesicles, implying that OVA (antigen) and CpG DNA (adjuvant) were ingested into the cell at the same time. This paper shows that OVA-SPG can be used as a CpG DNA carrier to induce antigenspecific immune responses.
INTRODUCTION Antigen-presenting cells (APCs), particularly dendritic cells, macrophages, and B-cells, play a crucial role in initiating immune responses. Generally, the immune responses are outcomes of the following events: foreign proteins are engulfed by APCs with phagocytosis and the fragments of the protein are presented on the surface of APCs as a major histocompatibility complex (MHC). Naı¨ve T cells bind MHC through T cell receptors, eventually, to induce differentiation of T cells into two cell subsets: T helper 1 (Th1) and T helper 2 (Th2) cells, depending on what cytokines are induced. Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) are known as an immune adjuvant to effectively induce Th1 response (1-4). After CpG DNAs are engulfed by APCs, they are recognized by toll-like receptor 9 (TLR-9) in phagosome-like vesicles to produce cytokines (5, 6). Among pro-inflammatory cytokines induced by CpG DNAs, interleukin (IL)-12 facilitates development of Th1 cells, and thus Th1 cells produce interferon γ (IFN-γ), which can suppress the differentiation of Th2 cells (7). Consequently, CpG DNAs can decrease Th2-mediated IgE production, which is a major cause * Corresponding author. Tel: +81-93-695-3294. Fax: +81-93-6953368. E-mail:
[email protected]. † The University of Kitakyushu. § Japan Science and Technology Agency (JST). ⊥ Department of Host Defense, Research Institute for Microbial Diseases, Osaka University. | The 21st Century COE, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University. ‡ Graduate School of Engineering, Kyushu University. # Center for Future Chemistry, Kyushu University.
of allergy if excessively secreted. Taking advantage of this response of CpG DNAs, they can be used as an antiallergy adjuvant. Especially, when phagocytosis-mediated antigen presentation is coupled with TLR-9-triggered events in the same cell, the resultant cytokines and co-stimulatory molecules should develop antigen-specific Th1 immunity. Therefore, to effectively induce this type of immunity, it is essentially important that CpG DNA and antigen are co-delivered to the same APC. The importance of the co-delivery has been proved by several groups using alum (8), cationic liposome (9), or bifunctional crosslinker (10, 11). However, there are some drawbacks in those methods. The most serious one is that CpG DNA is quickly degraded by nuclease under biological conditions. To protect CpG DNA, the terminal nucleotides are converted to phosphorothioate (12); however, phosphorothioate creates cytotoxicity and undesirable side effects (13, 14). Furthermore, cytotoxicity of cationic liposomes sometimes leads to serious side effects (15-17). We found that a natural polysaccharide called schizophyllan (SPG)1 forms a complex with single-stranded homo-polynucleotides (18). SPG is composed of a β-(1f3)-D-glucan main chain, and one β-(1f6)-D-glycosyl side chain links to the main chain every three glucose residues (Figure 1) (19). The complex can protect the bound DNA against nuclease-mediated hydrolysis or nonspecific binding to serum proteins. We have showed that these characteristics make it possible to use native phos1 Abbreviations: FITC, fluorescein isothiocyanate; FACS, fluorescence activated cell sorting; OVA, ovalbumin; F-OVA, FITC-labeled OVA; F-OVA-SPG, FITC-labeled OVA-SPG; CLSM, confocal laser scanning microscopy; ROX, rhodamine X; ROX-(dA)40, rhodamine X-labeled (dA)40; 1668-(dA)40, 5′-TCC ATG ACG TTC CTG ATG(dA)40-3′; SPG, schizophyllan.
10.1021/bc060070g CCC: $33.50 © 2006 American Chemical Society Published on Web 08/30/2006
Antigen-Conjugated Polysaccharide as CpG DNA Carrier
Figure 1. Repeating unit of schizophyllan.
phodiester DNAs for antisense therapy (20-22). Furthermore, SPG was selectively distributed in reticuloendothelial tissues such as liver, spleen lymph node, and bone marrow (23). On the basis of these findings, we have applied the SPG/DNA complex to a CpG DNA carrier. When the complex was added to macrophages or spleen cells, the secretion of IL-12 was enhanced over that with the naked dose. Moreover, when CpG DNA was complexed with SPG and intraperitoneally injected into mouse, the maximum secretion of IFN-γ was found to be twice as large as that invoked by the naked doses, and the secretion period was prolonged dramatically (24, 25). These excellent performances should be ascribed to both the stability of the SPG composite and the reticuloendothelial localization of SPG itself. In this paper, we synthesized an antigenconjugated SPG and examined the performance of the conjugate as a CpG DNA carrier using circular dichroism (CD), fluorescence activated cell sorting (FACS) analysis, and confocal laser scanning microscopy (CLSM).
EXPERIMENTAL PROCEDURES Materials. Mitsui Sugar Co., Ltd. (Japan) kindly supplied the SPG sample. The weight-average molecular weight (Mw) and the number of repeating units were found to be 1.5 × 105 and 231, respectively. Ovalbumin (OVA, a model antigen) was purchased from Sigma-Aldrich. As a CpG DNA, we adopted the sequence of 5′-TCC ATG ACG TTC CTG ATG-3′. To combine these sequences with SPG, we attached a (dA)40 tail at the 3′ end (26). Hereinafter, we denote this phosphodiester 5′-TCC ATG ACG TTC CTG ATG-(dA)40-3′ as 1668-(dA)40. For CD and CLSM, we used (dA)40 and rhodamine X-labeled (dA)40 (ROX-(dA)40), respectively. All DNA samples were synthesized at Hokkaido System Science (Hokkaido, Japan) and purified with high-pressure liquid chromatography. The lengths of the synthesized DNAs were confirmed with denatured PAGE. The fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco/BRL. Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Nissui Pharmaceutical Co., Ltd. Preparation of OVA-Conjugated SPG. We already established a method to introduce functional groups only to the side chain of SPG (26, 27). SPG (100 mg) was dissolved in water, sodium periodate (27 mg) was added to the solution, and the solution was stirred at 5 °C for 2 days to obtain an oxidized SPG bearing aldehyde groups. The oxidized SPG (1.25 mg/ mL), OVA (2.5 mg/mL), and sodium cyanoborohydride (100 mg) were dissolved into 0.1 M sodium bicarbonate, and the mixture was stirred at room temperature for 14 h. The OVAconjugated SPG (OVA-SPG) was purified by gel filtration (Sephacryl S200, Amersham), and the purity was examined with SDS-PAGE. The amount of OVA introduced to SPG was determined with a protein quantification kit (DOJINDO). Analysis of Gel Electrophoresis. OVA-SPG (or unmodified SPG) was dissolved in 97% DMSO and 3% H2O. The OVA-
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SPG solution (final concentration 0.67 mg/mL) was added to 1668-(dA)40 (final concentration 0.01 mg/mL) in a 30 mM TrisHCl (pH ) 7.0) buffer containing 0.15 M NaCl. The mixture was incubated at 5 °C for 2 days, and the formation of the complex was examined with agarose gel (2%) electrophoresis. The bands were visualized with GelStar (Amersham). Measurement of CD Spectra. OVA-SPG (or unmodified SPG) was dissolved in 97% DMSO and 3% H2O. The OVASPG solution (final concentration 0.67 mg/mL) was added to (dA)40 (final concentration 0.175 mg/mL) in a 30 mM TrisHCl (pH ) 7.0) buffer containing 0.15 M NaCl. The mixture was incubated at 5 °C for 2 days. CD was measured at 5 °C on a JASCO J-720WI spectrometer with a 1 mm cell. Cell Culture. Murine macrophage-like cells (J774.A1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The J774.A1 cells were maintained in DMEM supplemented with 10% FBS, containing a 1 wt % penicillin/ streptomycin mixture. The cell incubation was always carried out at 37 °C in fully humidified air containing 5 wt % CO2. Fluorescence Activated Cell Sorting (FACS) Analysis and Confocal Laser Scanning Microscopy (CLSM). FITC-labeled OVA-SPG was prepared for FACS and CLSM. FITC (72 µL of a 5 mg/mL solution) was added to 1 mL of OVA-SPG (4.4 mg/mL) in 0.1 M sodium bicarbonate (pH ) 9.0), and the mixture was stirred at 4 °C for 15 h. The mixture was purified with dialysis and gel filtration. FITC-labeled OVA was prepared by the same method. The amounts of OVA and FITC were determined with a protein quantification kit and the UV absorbance of FITC, respectively. The FITC/protein (F/P) molar ratios were 1.5 and 1.6 for F-OVA-SPG and F-OVA, respectively. For FACS, J774.A1 cells were plated in a 48-well dish (2 × 106 cells/mL; 200 µL/well) and allowed to attach to the wells at 37 °C for 24 h. The cells were washed twice with DMEM without FBS, and then 25 µg/mL (200 µL) OVA in F-OVASPG or F-OVA was added to the cells. The cells were incubated at 37 °C for 2 h, washed twice with PBS, harvested with a scraper, and fixed with 5% HCHO at 4 °C for 20 min. The number of the cells that had FITC was counted with an EPICS XL (BECKMAN COULTER). For CLSM, J774.A1 cells were plated in a glass-bottom dish (φ ) 12 mm) and allowed to attach to the dish at 37 °C for 24 h. The cells were washed twice with DMEM without FBS, and 200 µL of F-OVA-SPG/ ROX-(dA)40 complex or F-OVA was added to the cells at the concentration of 25 µg/mL OVA in F-OVA-SPG or F-OVA and 37.5 µg/mL ROX-(dA)40 in the complex. The cells were incubated at 37 °C for 2 h and washed twice with PBS and fixed with 5% HCHO containing 4′,6-diamidino-2-phenylindole (DAPI) at 4 °C for 20 min. The cells were observed with a Nikon fluorescence microscope (ECLIPS TE2000-U) with a Radiance2100 BioRad confocal scan unit attached.
RESULTS AND DISCUSSION Preparation of OVA-Conjugated SPG. OVA-SPG was synthesized as described in the Experimental Procedures section. Figure 2A shows the gel filtration chromatograms of a crude OVA-SPG (before purification, including a large amount of unconjugated OVA) and OVA itself. OVA has a large peak around 40-45 mL, and the crude OVA-SPG peak is overlaid with that of OVA. It can be seen that OVA-SPG conjugate was eluted at 25-30 mL as a shoulder. We carefully collected the shoulder ranging 25-28 mL. Figure 2B compares SDSPAGE patterns for OVA-SPG before and after the purification, as well as free OVA. Before the purification, two bands were observed (lane 4), corresponding the migrated and unmigrated bands to free OVA and OVA-SPG, respectively. After the purification, there was no appreciable band of free OVA,
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Figure 4. (A) Comparison of CD spectra for the mixture of OVASPG + (dA)40, unmodified SPG + (dA)40, SPG only, OVA-SPG only, and (dA)40 only measured at 5 °C in 30 mM Tris-HCl (pH ) 7.0) containing 0.15 M NaCl and 8.3% DMSO. (B) Temperature dependence of the CD intensity at 250 nm of the complexes and (dA)40 only. [unmodified SPG] ) 0.42 mg/mL, [OVA-SPG] ) 0.67 mg/mL, and [(dA)40] ) 0.175 mg/mL. Figure 2. (A) Gel filtration chromatograms of OVA-SPG and OVA. The shoulder ranging 25-28 mL was collected. (B) Analysis of OVASPG by 8% SDS-PAGE: lane 1, a molecular weight marker; lane 2, OVA only; lane 3, SPG only; lane 4, the crude sample examined in the gel filtration; lane 5, the purified sample obtained from the shoulder.
Figure 3. Confirmation of complex formation between OVA-SPG and 1668-(dA)40 by 2% agarose gel electrophoresis stained with GelStar: lane 1, 1668-(dA)40 only; lane 2, mixture of unmodified SPG and 1668-(dA)40; lane 3, mixture of OVA-SPG and 1668-(dA)40; [unmodified SPG] ) 0.42 mg/mL, [OVA-SPG] ) 0.67 mg/mL, and [1668-(dA)40] ) 0.01 mg/mL.
Figure 5. FACS profiles of F-OVA or F-OVA-SPG-treated macrophage-like cells (J774.A1). OVA (25 µg/mL) in F-OVA or F-OVA-SPG was added to the cells in DMEM without FBS at 37 °C for 2 h. Black line, blue line, and red line indicate untreated cells, F-OVA-treated cells, and F-OVA-SPG-treated cells, respectively.
indicating that the purification removed all free OVA. We determined that 0.4 mg of OVA per 1 mg of OVA-SPG was introduced, meaning that two OVA (43 kDa) molecules were attached to one SPG (Mw ) 150 000) molecule. DNA Binding Ability of OVA-SPG. Figure 3 shows agarose gel electrophoresis when 1668-(dA)40 only, SPG/1668-(dA)40, and OVA-SPG/1668-(dA)40 were applied. The 1668-(dA)40 migrated into the gel. On the other hand, the complexed 1668(dA)40 with unmodified SPG stayed at the well and never moved to show a retarded band, which is evidence of the complex formation (lane 2) (28). The retarded band at the well was observed in lane 3 in the same way as in lane 2, indicating that OVA-SPG formed a complex with 1668-(dA)40. To confirm the complexation, CD spectra were measured (Figure 4A). In this case, for convenience, we used (dA)40 instead of 1668(dA)40. OVA-SPG itself and SPG itself were not CD-active, showing no spectrum at 240-320 nm. When OVA-SPG was added to (dA)40, a negative band at 250 nm disappeared, and a new negative band at 275 nm appeared. The band at 280 nm in (dA)40 itself seemed to red-shift and increase in intensity upon complexation with OVA-SPG. These spectral changes completely agree with those of the SPG/(dA)40 complex (29),
indicating that the attachment of two OVA molecules to SPG does not interfere in the complexation ability at all. Figure 4B plots the CD intensity at 250 nm of OVA-SPG/ (dA)40 complex against temperature, compared with SPG/(dA)40 and (dA)40. Owing to the presence of the complexes, 250 nm CD intensities for OVA-SPG/(dA)40 and SPG/(dA)40 are larger than that of (dA)40 at low temperatures. They merge into that of (dA)40 above 55 °C, indicating that the dissociation of the complex takes place at this temperature. The melting (or dissociation) curves are almost the same between the two complexes. These results indicate that OVA-SPG can form a thermally stable complex with DNA in the same manner as SPG/ DNA complexes. The morphological (30) and fundamental thermodynamic properties (18, 28) of SPG/nucleic acid complexes have been well studied, and the similarity in CD spectrum between SPG/DNA and OVA-SPG/DNA complexes implies that the basic properties of the complex are not changed after conjugation of OVA. Ingestion and Distribution of OVA-SPG and Complexed DNA. Figure 5 compares the FACS profiles between F-OVA and F-OVA-SPG when these were added to J774.A1. The number of cells that had ingested FITC was increased when F-OVA-SPG was added, compared with F-OVA. This result
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most of F-OVA and ROX-(dA)40 seem not to localize at the same vesicles, indicating that both objects were ingested independently. Figure 6B is the CLSM image when the F-OVA-SPG/ROX-(dA)40 complex was added. F-OVASPG and ROX-(dA)40 are distributed heterogeneously within cytosol in the same manner as Figure 6A. In contrast to the previous image, most images are yellow, indicating that a large number of F-OVA-SPG and ROX-(dA)40 are colocalized in the same vesicles. In addition, there seemed to be a quantitative difference in the ingested CpG DNA between photos A and B in Figure 6. OVA-SPG induces more uptake of CpG DNA than SPG itself. Presumably, an unknown uptake mechanism for OVA as an antigen might be responsible for this increment. The present results imply that OVA (antigen) and CpG DNA (adjuvant) were ingested at the same time and delivered to the same vesicles when CpG DNA and OVA-SPG were complexed. Therefore, we can speculate that the present complex can antigen-specifically induce a TLR-9-dependent Th1 immune response.
CONCLUSIONS An OVA-conjugated SPG was synthesized, and the characterization of the conjugate was carried out. Composition analysis showed that two OVA molecules are attached to one SPG molecule, and agarose gel electrophoresis and CD proved that the conjugate can form a complex with 1668-(dA)40. When the FITC-labeled conjugate was added to J774.A1 cells, the number of cells that had ingested FITC was increased compared with OVA itself, suggesting that the cells ingested the conjugate by SPG/Dectin-1 recognition. CLSM showed that the ingested OVA and DNA were in the same vesicles. We believe that antigen-conjugated SPG is applicable as a carrier to induce an antigen-specific and TLR-9-dependent Th1 immune response.
ACKNOWLEDGMENT This work is financially supported by JST SORST and ERATO programs and Grant-in-Aid for Scientific Research (16350068 and 16655048).
LITERATURE CITED
Figure 6. Intracellular localization of (A) a mixture of unmodified SPG/ROX-(dA)40 and F-OVA and (B) the complex made from F-OVA-SPG and ROX-(dA)40 in J774.A1 cells. The blue, green, and red colors correspond to DAPI (nucleus), FITC (OVA), and ROX (DNA), respectively. The yellow color is the outcome of the merge between FITC and ROX, which is only observed for the complex.
indicates that the presence of SPG enhanced the cellular ingestion. It has been reported that β-1,3-glucans, especially for zymosan and other cell-wall glucans, are recognized by a receptor called Dectin-1 (31) and are internalized by phagocytosis (32). Increased uptake of OVA-SPG might be ascribed to the receptor-mediated uptake for SPG. By use of CLSM, we examined the intracellular distribution for the OVA-SPG/ROX-(dA)40 complex compared with a mixture of F-OVA and SPG/ROX-(dA)40. Figure 6A shows the CLSM image when a mixture of F-OVA and the unmodified SPG/ROX-(dA)40 complex were added to the J774.A1 cells, where F-OVA and the SPG/ROX-(dA)40 complex do not form a complex. The image A shows that F-OVA and ROX-(dA)40 are distributed heterogeneously within the cytosol. However,
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