Nontoxic B Subunit of Cholera Toxin Cpg Oligodeoxynucleotide Linked

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Novel Immunostimulatory Agent Based on CpG Oligodeoxynucleotide Linked to the Nontoxic B Subunit of Cholera Toxin

This information is current as Jenni Adamsson, Marianne Lindblad, Annika Lundqvist, of September 28, 2021. Denise Kelly, Jan Holmgren and Ali M. Harandi J Immunol 2006; 176:4902-4913; ; doi: 10.4049/jimmunol.176.8.4902 http://www.jimmunol.org/content/176/8/4902 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Novel Immunostimulatory Agent Based on CpG Oligodeoxynucleotide Linked to the Nontoxic B Subunit of Cholera Toxin1

Jenni Adamsson,* Marianne Lindblad,* Annika Lundqvist,* Denise Kelly,† Jan Holmgren,* and Ali M. Harandi2*

In this study, we report the development of a novel, rationally designed immunostimulatory adjuvant based on chemical conjugation of CpG oligodeoxynucleotide (ODN) to the nontoxic B subunit of cholera toxin (CTB). We demonstrate that the immunostimulatory effects of CpG can be dramatically enhanced by conjugation to CTB. Thus, CpG ODN linked to CTB (CTB-CpG) was shown to be a more potent stimulator of proinflammatory cytokine and chemokine responses in murine splenocytes and human PBMCs than those of CpG ODN alone in vitro. The presence of CpG motif, but not modified phosphorothioate ODN Downloaded from backbone, was found to be critical for the enhanced immunostimulatory effects of CTB-CpG. Our mode-of-action studies, including studies on cells from specifically gene knockout mice suggest that similar to CpG, CTB-CpG exerts its immunostimulatory effects through a TLR9/MyD88- and NF-␬B-dependent pathway. Surprisingly, and as opposed to CpG ODN, CTB-CpG-induced immunity was shown to be independent of endosomal acidification and resistant to inhibitory ODN. Furthermore, preincubation of CTB-CpG with GM1 ganglioside reduced the immunostimulatory effects of CTB-CpG to those of CpG ODN alone. Interest- ingly, conjugation of CpG ODN to CTB confers an enhanced cross-species activity to CpG ODN. Furthermore, using tetanus http://www.jimmunol.org/ toxoid as a vaccine Ag for s.c. immunization, CTB-CpG markedly enhanced the Ag-specific IgG Ab response and altered the specific pattern of Ab isotypes toward a Th1 type response. To our knowledge, CTB is the first nontoxic derivative of microbial toxins discovered that when chemically linked to CpG remarkably augments the CpG-mediated immune responses. The Journal of Immunology, 2006, 176: 4902–4913.

nmethylated CpG dinucleotides with particular sequence oxynucleotide (ODN)3-containing CpG motifs. The use of CpG context (CpG motifs) occur at high frequency in micro- ODN as a Th1-tilting immunostimulator/adjuvant either singly or bial DNA and stimulate the mammalian immune system in combination with various Ags for induction of both systemic U by guest on September 28, 2021 (1). Cellular responses to CpG DNA require initial internalization and mucosal immunity in experimental animals has been docu- of the DNA followed by endosomal acidification. CpG binds to mented (2, 3). TLR9 located intracellularly on endosomal membranes. TLR9 sig- The immunostimulatory effects of CpG ODNs are dependent on naling proceeds through recruitment of MyD88 leading to activa- a number of factors, including DNA sequence, nature of the back- tion of several kinases, including IL-1R-associated kinase and bone, and the presence of species-specific motifs. Although phos- TNFR-associated factor 6. The signal cascade culminates in the phodiester (PO) CpG ODNs are poor inducers of immune re- activation of NF-␬B, AP-1, and members of the MAPK family that sponses, CpG ODNs with a modified phosphorothioate (PS) trigger rapid induction of various proinflammatory and Th1-polar- backbone possess potent immunostimulatory effects (2, 4). Mice izing cytokines and chemokines, up-regulation of costimulatory and humans respond preferentially to different CpG motifs. In molecules on APCs, and activation of B cells for proliferation, mice, the optimal motif consists of a central unmethylated CpG IL-6 secretion, and Ab production (2). The immunostimulatory dinucleotide flanked by two 5Ј purines and two 3Ј pyrimidines features of bacterial DNA can be mimicked by synthetic oligode- such as GACGTT (e.g., CpG ODN 1826) (5, 6). For humans, GTCGTT in multiple copies, separated by TT dinucleotides (e.g., CpG ODN 2006) is optimal (7). Efforts have been made to augment the immunostimulatory ef- *Department of Medical Microbiology and Immunology, Go¨teborg University Vac- fects of CpG ODNs. Thus, adsorption of CpG ODN to the surface cine Research Institute (GUVAX), Go¨teborg University, Go¨teborg, Sweden; and †Gut Immunology Group, Rowett Research Institute, Bucksburn, Aberdeen, United of cationic poly(D,L-lactide-co-glycolide) microspheres was shown Kingdom to induce enhanced secretion of IFN-␥ and IFN-␣ from human Received for publication July 14, 2005. Accepted for publication January 23, 2006. PBMC compared with CpG ODN alone (8). Furthermore, a syn- The costs of publication of this article were defrayed in part by the payment of page ergistic adjuvant activity was reported when CpG ODN was mixed charges. This article must therefore be hereby marked advertisement in accordance with cholera toxin (CT) or Escherichia coli heat-labile enterotoxin with 18 U.S.C. Section 1734 solely to indicate this fact. (LT); however, a mixture of CpG ODN and nontoxic derivatives of 1 This work was supported by the Swedish Research Council Medicine, the Swedish International Development Cooperation Agency, the Swedish Medical Society Re- CT and LT, i.e., the B subunit of CT (CTB) and the mutant of LT search Foundation against AIDS, and Scottish Executive for Environmental and Rural Affairs Department. GUVAX is supported by the Knut and Alice Wallenberg Foundation. 2 Address correspondence and reprint requests to Dr. Ali M. Harandi, Institute of 3 Abbreviations used in this paper: ODN, oligodeoxynucleotide; PO, phosphodiester; Biomedicine, Department of Microbiology and Immunology, Go¨teborg University PS, phosphorothioate; CT, cholera toxin; LT, Escherichia coli heat-labile enterotoxin; Vaccine Research Institute (GUVAX), Sahlgrenska Academy at Go¨teborg Uni- CTB, B subunit of CT; TGN, trans-Golgi network; ER, endoplasmic reticulum; TT, versity, Go¨teborg, Sweden. E-mail address: [email protected] tetanus toxoid; EU, endotoxin unit; WMN, wortmannin.

protein derivative LTK63, had no synergistic effects on the mag- either a PS or a PO backbone. The ODNs were purchased from Cybergene nitude of the immune response (9–11). (Novum Research Park) and Operon. ODNs were tested for endotoxin, and Ͻ CT is an AB5 hexameric protein responsible for the severe chol- the LPS contents were 0.03 endotoxin units (EU)/ml. rCTB protein and TT were provided by SBL Vaccines. All other re- era diarrhea caused by Vibrio cholera (12). CT consists of five agents were purchased from Sigma-Aldrich unless stated otherwise. identical B subunits and a single A subunit. Cell recognition and binding of CT are conducted by the B pentamer via specific bind- Conjugation of CpG ODN to CTB ing to the ganglioside GM1 receptor, a glycosphingolipid found The conjugation of CpG ODN to CTB was performed by mixing thiolated ubiquitously on the surface of mammalian cells. High-affinity in- ODN with maleimide-activated CTB at room temperature overnight, fol- teraction of CTB with GM1 ganglioside receptors in lipid rafts of lowed by purification by gel filtration through Superdex 200 (Amersham the plasma membrane triggers holotoxin internalization into Biosciences). The high molecular peak was collected and protein concentration was determined using a protein assay (micro BCA; Pierce), with smooth endocytic vesicles that are targeted to the trans-Golgi net- CTB as a reference protein. Concentration of nucleic acid was determined work (TGN) and the endoplasmic reticulum (ER). The enzymati- by measuring the absorbance at 260 nm. The CTB-ODN conjugates were cally active A subunit is then directed to the interior of the target analyzed by SDS-PAGE and CTB and CpG ODN in the conjugate were cell where it causes an increase in the intracellular level of cAMP visualized with Coomassie Blue (Sigma-Aldrich) and SYBR Green (Mo- lecular Probes), respectively. The GM1-binding activity of CTB-ODN con- (13). Several reports indicate that the immunogenicity of protein jugates was confirmed by a solid-phase GM1 ELISA (15). The LPS con- Ags can be enhanced when they are conjugated to CTB rather than tents of ODN-CTB conjugates were 0.1 EU/ml. simply mixed with it. CTB, as opposed to CT, can be safely ad- ministered to humans (14). Animals

In this study, we investigated the possibility of enhancing im- Six- to 10-wk-old mice were used for all experiments. C57BL/6 mice were Downloaded from munostimulatory and adjuvant effects of CpG by combining the purchased from M&B. C3H/HeN and C3H/HeJ mice were purchased from Ϫ/Ϫ Ϫ/Ϫ immunostimulatory effect of CpG with the specific cell binding Charles River Laboratories. MyD88 (16) and TLR9 (17) mice on C57BL/6 background were originally from Prof. S. Akira’s laboratory (gift and intracellular shuttling ability of CTB. To achieve this goal, we from Prof. M. J. Wick, Department of Clinical Immunology, Go¨teborg chemically linked synthetic CpG ODN to rCTB protein. We report University, Go¨teborg, Sweden). Animals were housed in microisolators here that the immunostimulatory effects of CpG can be dramati- under specific pathogen-free conditions at the Experimental BioMedicine animal facility (Sahlgrenska Academy, Go¨teborg University, Go¨teborg, cally potentiated by conjugation to CTB. Thus, CpG linked to, but http://www.jimmunol.org/ Sweden). All experiments were performed with the approval from the Eth- not simply admixed with, CTB is a more potent stimulator of cy- ical Committee for Laboratory Animals (Go¨teborg, Sweden). tokine and chemokine responses in murine splenocytes and human PBMCs than CpG ODN in vitro. Interestingly, conjugation of PO In vitro human proliferation assays CpG ODN with little immunostimulatory effect to CTB converts it Human PBMC were isolated from buffy coats provided by the blood bank into a potent stimulator of cytokine and chemokine responses in of the Sahlgrenska Hospital (Go¨teborg, Sweden). PBMCs were prepared vitro. We also report a series of studies that analyze the mode of from buffy coats by Ficoll-Hypaque (Amersham Biosciences) density gra- action of CTB-CpG. These studies suggest that similar to CpG, dient centrifugation. PBMCs were suspended in basal Iscove’s medium (Biochrom) supplemented with 2 mM L-glutamine (VWR), 50 ␮M 2-ME, CTB-CpG exerts its immunostimulatory effects through a TLR9/ 100 ␮g/ml gentamicin, and 10% heat-inactivated FCS (referred to as MYD88- and NF-␬B-dependent pathway. Surprisingly and as op- Iscove’s complete medium) and seeded in triplicates in 96-well flat-bottom by guest on September 28, 2021 posed to CpG ODN alone, CTB-CpG-induced immunity was in- plates (Nunc) and stimulated with different concentrations of ODNs, re- dependent of endosomal acidification and also resistant to combinant CTB, CTB ϩ CpG, CTB-ODNs. Supernatants were collected at inhibitory ODN. Preincubation of CTB-CpG with GM1 ganglio- different time points and assayed for cytokines and chemokines contents. On day 3, cells were pulsed with 1 ␮Ci of [3H]thymidine (Amersham side reduced the immunostimulatory effects of CTB-CpG to those Biosciences) for the last 6–8 h of culture. The cellular DNA was harvested of CpG ODN alone. Furthermore, we could show that the conju- on glass fiber (Wallac) and assayed by liquid scintillation counting. gation of CpG ODN to CTB can override the species-specificity of In vitro murine proliferation assays CpG ODN. Finally, using tetanus toxoid (TT) as a vaccine Ag for s.c. immunization of mice, CTB-CpG has been shown to markedly Spleens were removed from anesthetized animals under aseptic conditions augment the induction of Ag-specific IgG Ab response and to tilt and homogenized. Splenocytes were resuspended in the Iscove’s complete medium, and incubated in the presence of CpG ODN, rCTB, a mixture of the specific pattern of Ab isotypes toward a Th1 type response, CTB and CpG or CTB-CpG conjugate. Supernatants were collected and suggesting that CTB-CpG could be used as a potent immune ad- examined for their contents of cytokines and chemokines. juvant. To our knowledge, CTB is the first nontoxic derivative of For mode of action experiments, cell suspensions of murine splenocytes microbial toxins discovered that when chemically linked to CpG were pretreated with chloroquine (2 or 4 ␮g/ml), quinacrine (0.1 or 0.4 ␮ ␮ ␮ dramatically augments the CpG-mediated immune responses. This g/ml), wortmannin (2.5 g/ml), or gliotoxin (0.25 g/ml) for2hat37°C, followed by cultivation in the presence of CpG ODN (0.3 ␮g/ml), CTB, observation warrants further exploration of the impact of the new CTB ϩ CpG or CTB-CpG. In some experiments, inhibitory ODN 1502 (1, class of immunostimulator/adjuvant based on CpG ODN coupled 3, 9 ␮g/ml) or polymyxin B (10 ␮g/ml) was added to the cell culture at the to CTB for inducing more potent immune responses. same time as reagents. For the GM1 receptor blocking experiment, the reagents were preincubated with GM1 ganglioside (2.5 or 10 ␮g/ml) for 30 min at 37°C before Materials and Methods being added to the C57BL/6 splenocyte culture. ODN and reagents Cytokine and chemokine quantification CpG ODN 2006, a 24-mer that contains four copies of a CpG motif (TCG Concentrations of cytokines and chemokines in pooled culture supernatants TCG TTT TGT CGT TTT GTC GTT), was used for human in vitro studies. were analyzed using cytokine/chemokine Duoset, fluorokine ELISA, or Human ODN IMTO22, a 24-mer with no CpG motifs (TGC TGC AAA matched Ab pairs (R&D Systems), according to the manufacturer’s AGA GCA AAA GAG CAA), was used as a control. For mouse studies, recommendations. CpG ODN 1826, a 20-mer containing two copies of a CpG motif (TCC ATG ACG TTC CTG ACG TT) with potent immunostimulatory effects on NF-␬B EMSA the murine immune system, was used. The mouse ODN, a 20-mer with no CpG motifs TCC AGG ACT TCT CTC AGG TT, was used as a control. Human PBMCs were stimulated with CpG ODN, CTB, or CpG-CTB for Mouse ODN 1502, a 20-mer, which contains two copies of a GpC motif periods up to 2 h. Following stimulation, cells were harvested into ice-cold (GpC ODN) (GAG CAA GCT GGA CCT TCC AT), was also used. All PBS and lysed in hypotonic buffer (0.125% Igepal CA-630, 5 mM HEPES

ODNs were used with complete PS backbone. CpG 2006 was used with (pH 7.9), 10 mM KCL, 1.5 mM MgCl2). Following centrifugation 4904 NOVEL IMMUNOSTIMULATORY AGENT BASED ON CpG DNA LINKED TO CTB

(13,000 ϫ g, 1 min, 4°C), nuclear pellets were resuspended in hypertonic PBMCs. To this end, human PBMCs were cultured in the presence lysis buffer (0.25% Igepal CA-630, 5 mM HEPES (pH 7.9), 25% glycerol, of increasing concentrations of CTB or CpG 2006, either alone or 500 mM MgCl , 02 mM EDTA) for 2 h at 4°C with constant mixing. 2 in mixed (CTB ϩ CpG 2006) or conjugated (CTB-CpG 2006) Nuclear extract supernatants were recovered by centrifugation (13,000 ϫ g, 10 min, 4°C), aliquoted and stored at Ϫ80°C before EMSAs. NF-␬B form. Supernatants of the cells were collected at different time DNA-binding activities of nuclear extracts were determined using 4 ␮gof points and examined for proinflammatory cytokines and nuclear protein incubated with [␥-32P]ATP NF-␬B double-stranded con- chemokines. ␮ sensus oligonucleotide (Promega). For supershift analysis, 1 gofp65 Basal levels of MIP-1␣ and MIP-1␤ were observed in the su- (Rel A) Ab (Santa Cruz Biotechnology) was added to the nuclear extract Ϯ Ϯ mix. Samples were resolved using 5% (w/v) nondenaturing polyacrylamide pernatants of the human PBMCs at 24 h (1350 400 and 9779 gels. Gels were dried and NF-␬B DNA-binding protein complexes were 929 pg/ml/million cells, respectively). Incubation of the cells with visualized using a Fujifilm LAS-1000 image analyzer. CTB did not induce any MIP-1␣ or MIP-1␤ productions over those of untreated cells at any concentrations or time points ex- Immunization amined (Fig. 1, A–C). Incubations of PBMCs with CpG 2006 (3 Groups of three female C57BL/6 mice were immunized s.c. with TT (10 ␮g/ml) induced a 3- and 5-fold increases in MIP-1␣ and MIP-1␤ ␮ ␮ ␮ g) alone or mixed with CTB (18 g), CpG ODN (5 g), or CTB-CpG productions over those of cells alone at 24 h, respectively, that (CTB: 18 ␮g, CpG: 5 ␮g) three times with 7–10 day intervals. Two weeks after the last immunization, mice were sacrificed and blood samples (from declined to the basal level within 48 h. No additional increase was axillary plexus) and spleens were collected. observed in the supernatants of the cells treated with CTB ϩ CpG 2006. However, CTB-CpG 2006 (3 ␮g/ml) induced robust, dose- Ab measurements dependent induction of MIP-1␣ (182-fold increase) and MIP-1␤

Low-binding 96-well plates (Nunc) were coated with TT at a concentration (46-fold increase) responses at 24 h and remained elevated for 72 h Downloaded from of 2 ␮g/ml in carbonate buffer at 4°C overnight. Plates were blocked with (Fig. 1, A–F). 4% milk (Semper) for2hat37°C. Sera were serially diluted in 4% milk IL-6 production followed the same pattern as those observed for and incubated for1hat37°C. Bound Abs were detected using HRP- ␣ ␤ ϩ conjugated goat-anti-mouse IgG, IgG1, or IgG2c (Jackson ImmunoRe- MIP-1 and MIP-1 . Thus, CpG 2006 and CTB CpG 2006 search Laboratories). The plates were developed with o-phenylene-diamine induced comparable levels of IL-6 response in human PBMCs. dihydrochloride and read at 450 nm. The sample IgG titer was deﬁned as CTB-CpG 2006, in contrast, induced a very strong IL-6 response

the sample dilution giving an OD value of 0.4 above the background value. in a dose-dependent manner that remained moderately high for http://www.jimmunol.org/ 72 h (Fig. 1, G–I). Results As tabulated in Table I, CpG 2006 (3 ␮g/ml) induced moderate CTB-CpG as a potent stimulator of cytokines and chemokines in levels of IL-1␤ (4-fold increase over untreated cells) and MCP-1 human PBMCs (2-fold increase over untreated cells) productions in human PB- We sought to examine whether conjugation of CpG ODN to CTB MCs. CpG 2006 did not induce any appreciable change in the level would enhance immunostimulatory effects of CpG ODN in human of IL-8 in human PBMCs. CTB-CpG 2006 induced relatively by guest on September 28, 2021

FIGURE 1. Comparison of CpG ODN, CTB, CTB ϩ CpG, and CTB-CpG for their ability to induce MIP-1␣, MIP-1␤, and IL-6 responses in human PBMCs. A–I, Human PBMCs were incubated with different concentrations of CTB or CpG 2006, either alone or in the mixed or conjugated form. Cell supernatants were collected at 24 h (A, D, and G), 48 h (B, E, and H) and 72 h (C, F, and I) and examined for MIP-1␣, MIP-1␤, and IL-6 contents by ELISA. Data shown are representative of ﬁve independent experiments and are expressed as mean of duplicate ELISA samples Ϯ SEM. The Journal of Immunology 4905

Table I. The effect of CpG and CTB-CpG on induction of proinﬂammatory cytokine and chemokines as well as proliferative responses in human PBMCsa

Untreated Cells CpG 2006 CTB-CpG 2006

IL-1␤ 0.2 Ϯ 0 0.8 Ϯ 0.04 134 Ϯ 0.5 IL-8 218 Ϯ 6.2 205 Ϯ 38 993 Ϯ 12 MCP-1 217 Ϯ 1.2 530 Ϯ 25 1951 Ϯ 1 Proliferative response 112 Ϯ 11 7820 Ϯ 378 5545 Ϯ 117

a Human PBMCs were incubated with CpG 2006 (3 ␮g/ml) or CTB-CpG 2006 (CpG content: 3 ␮g/ml). The cell supernatants were analyzed for IL-1␤, IL-8, and MCP-1 (24-h supernatants). Cell proliferative responses were examined after 72 h as described in Materials and Methods. Data shown are representative of two independent experiments and are expressed as nanograms per milliliter per million cells (for IL-1␤, IL-8, and MCP-1) and cpm (for proliferative response). strong IL-1␤ (167-fold increase over CpG 2006), IL-8 (5-fold in- MIP-1␤, and IL-6 responses with similar kinetic, but did not reach crease over CpG 2006), and MCP-1 (4-fold increase over CpG the levels induced by CTB-CpG PS (Fig. 2, D–F). Interestingly, 2006) responses in human PBMCs (Table I). Neither CpG 2006 incubation of PBMCs with either CpG PO or CTB-CpG PO did nor CTB-CpG 2006 elicited any appreciable change in the levels not result in any appreciable levels of proliferative response (data of IFN-inducible protein-10 and RANTES in human PBMCs (data not shown). These data demonstrate that linkage of CpG PO to

not shown). CTB converts a molecule with little inherent immunostimulatory Downloaded from The effects of CTB or CpG 2006, either alone or in the mixed effect into a potent stimulator of proinﬂammatory cytokine and (CTB ϩ CpG 2006) or conjugated (CTB-CpG 2006) form on in- chemokine responses. duction of proliferative response in human PBMCs were examined. CTB alone did not induce any proliferative response in hu- Conjugation of optimal mouse CpG to CTB enhances the man PBMCs. Treatment of the cells with CpG 2006, in contrast, immunostimulatory effects of mouse CpG in vitro

gave rise to a strong proliferative response (Table I). However, We investigated the immunostimulatory effects of optimal mouse http://www.jimmunol.org/ treatment of the cells with mixture of CTB ϩ CpG 2006 (data not CpG linked to CTB in murine splenocytes. Murine splenocytes shown) or CTB-CpG 2006 did not confer a further increase in were cultivated in the presence of increasing concentrations of proliferative response over that of CpG 2006 (Table I). Taken to- CTB, CpG 1826, CTB ϩ CpG 1826, or CTB-CpG and superna- gether, these data indicate that conjugation of CTB to optimal hu- tants were collected for cytokine/chemokine analysis and cells as- man CpG 2006 can dramatically potentiate the stimulatory effect sessed for proliferation after 72 h. CTB did not induce any MIP- of optimal human CpG on proinflammatory cytokine and chemo- 1␣, MIP-1␤, and IL-6 productions in murine spleen cells at any kine responses in vitro without affecting cell proliferation to an concentrations examined (data not shown). Although concentra- appreciable extent. tion of 0.1 ␮g/ml CpG did not induce any appreciable levels of these cytokines, incubation of the splenocytes with 0.3 ␮g/ml by guest on September 28, 2021 CpG motif is critical for the enhanced immunostimulatory effects (for MIP-1␣ and MIP-1␤)or1␮g/ml (for IL-6) of CpG resulted of CTB-CpG in MIP-1␣, MIP-1␤, and IL-6 responses. The highest MIP-1␣ and Next, we sought to ascertain the relative importance of the CpG MIP-1␤ responses were observed when 1 ␮g/ml CpG was used. motif in the immunostimulatory effects of CTB-CpG. To this end, Splenocytes treated with CTB ϩ CpG produced comparable levels human PBMCs were cultured in the presence of increasing con- of MIP-1␣, MIP-1␤, and IL-6 to those observed for CpG-treated centrations of control ODN IMTO22 (a control GpC ODN for cells. Interestingly, as little as 0.1 ␮g/ml CTB-CpG elicited strong CpG 2006 lacking the CpG motif), CTB-IMTO22, CpG 2006, or MIP-1␣, MIP-1␤, and IL-6 responses, which were comparable CTB-CpG 2006. Incubation of the cells with ODN IMTO22 in- with those observed for 1 ␮g/ml CpG. The CTB-CpG induced duced low levels of MIP-1␣, MIP-1␤, and IL-6. At the highest dose-dependent increases in cytokines and chemokines that concentration (9 ␮g/ml), CTB-IMTO22 induced slight increases in reached maximal levels at the concentration of 1 ␮g/ml (Fig. 3, MIP-1␣, MIP-1␤, and IL-6 productions over those of IMTO22- A–C). Consistent with human data, CTB did not induce a prolif- treated cells. As expected, CTB-CpG 2006 induced a dramatic erative response in the murine splenocytes. As expected, CpG in- increase in MIP-1␣, MIP-1␤, and IL-6 responses over those of duced high levels of proliferative response in a dose-dependent CpG 2006 (Fig. 2, A–C). Neither IMTO22 nor CTB-IMTO22 in- manner. CTB ϩ CpG did not give rise to any additional increase duced any significant proliferative response in human PBMCs in proliferative response over those of CpG. CTB-CpG, in contrast, (data not shown). These results imply that CpG motif is a critical induced a significantly stronger splenocyte proliferative response component for immunostimulatory effects of CTB-CpG conjugate. compared with those of CpG (Fig. 3D). To exclude the possibility that chemical modifications of CTB Linking of CpG with PO backbone to CTB augments its (maleimide activation) and CpG ODN (thiolation) are responsible immunostimulatory effect for the enhanced immunostimulatory effects of CTB-CpG, murine Next, we sought to examine whether immunostimulatory effects of splenocytes were cultivated with CTB, maleimide CTB, CpG CpG with PO backbone (CpG PO) can also be enhanced by con- ODN or thiolated CpG ODN. As illustrated in Fig. 3, E and F, the jugation to CTB. To this end, human PBMCs were incubated with chemical modifications of CTB and CpG ODN did not give rise to increasing concentrations of CpG 2006 PS, CpG 2006 PO, CTB ϩ enhanced MIP-1␣ and proliferative responses over those of CTB CpG PS/CpG PO, CTB-CpG PS, or CTB-CpG PO. Incubation of and CpG, respectively. Thus, the chemical modifications of CTB PBMCs with CpG 2006 PS induced low amounts of MIP-1␣, MIP- and CpG ODN are not responsible for the enhanced immuno- 1␤, and IL-6, whereas CpG PO did not induce any appreciable stimulatory effects of CTB-CpG. levels of these cytokines. Incubation of PBMCs with CTB-CpG PS To examine the importance of CpG motif in the CTB-CpG- resulted in robust MIP-1␣, MIP-1␤, and IL-6 responses that stayed induced immune response, we tested the immunostimulatory ef- markedly elevated for 72 h. CTB-CpG PO induced strong MIP-1␣, fects of a GpC containing ODN (GpC ODN) linked to CTB in 4906 NOVEL IMMUNOSTIMULATORY AGENT BASED ON CpG DNA LINKED TO CTB Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. The presence of CpG motif, but not phosphorothioate backbone, is crucial for CTB-CpG-induced proinflammatory cytokine and chemokine responses. A–C, Human PBMCs were incubated in triplicates with different concentrations of CpG 2006, IMTO22 (a non-CpG containing control ODN), CTB-CpG 2006, IMTO22, CTB-IMTO22. Cell supernatants were analyzed after 24 h for MIP-1␣, MIP-1␤, and IL-6 levels by ELISA. D–F, Human PBMCs were cultured with increasing concentrations of CpG 2006 with PS or PO backbone, either alone or in the conjugated form with CTB (CTB-CpG 2006). Data shown are representative of two independent experiments and are expressed as mean of duplicate ELISA samples Ϯ SEM. murine splenocytes. Murine splenocytes were cultured in the pres- ganglioside. Murine splenocytes were cultured with CpG 1826, ence of GpC ODN, CTB-GpC, CpG 1826, or CTB-CpG 1826. CTB-CpG1826, GM1-treated CpG 1826, or GM1-treated CTB- GpC ODN induced virtually no MIP-1␣, MIP-1␤, and prolifera- CpG 1826 followed by analysis of proliferative and cytokine/che- tive responses in murine splenocytes. CTB-GpC induced a mar- mokine responses. As depicted in Fig. 5, splenocytes cultivated ginal increase in the chemokine response over those of GpC ODN with CpG or GM1-treated CpG produced similar levels of MIP-1␣. (Fig. 4, A and B). CTB-GpC elicited a more potent proliferative Likewise, comparable levels of proliferative response were ob- response compared with that of GpC ODN; however, the level of served in splenocytes incubated with CpG or GM1-treated CpG. CTB-GpC-induced proliferative response did not reach that of However, pretreatment of CTB-CpG with GM1 dramatically re- CTB-CpG (Fig. 4C). Taken together, these results indicate that the duced immunostimulatory effects of CTB-CpG. Although CTB- immunostimulatory effect of optimal mouse CpG can be enhanced CpG induced strong MIP-1␣, MIP-1␤, IL-6, and proliferative re- by conjugation to CTB and that such conjugation can dramatically sponses in murine splenocytes, the GM1 treatment of CTB-CpG lower the concentration of CpG needed for stimulating immune significantly reduced the magnitude of the observed immune re- responses in murine splenocytes. sponses (Fig. 5 and data not shown). These results suggest that the GM1 receptor-binding property of CTB-CpG is required for its Incubation with GM1 ganglioside diminishes the enhanced immunostimulatory effects. immunostimulatory effects of CTB-CpG Given the importance of the GM1 receptor for the cellular uptake Blocking of endosomal acidification does not abolish CTB-CpG- of CTB, we examined whether preincubation of CTB-CpG with induced immune response GM1 ganglioside could influence the immunostimulatory effects of Previous studies have shown that endosomal acidification is a crit- CTB-CpG. To this end, CpG 1826 and CTB-CpG 1826 were pre- ical step for the immunostimulatory effects of CpG DNA. To de- incubated at 37°C for 30 min in the presence or absence of GM1 termine whether the CTB-CpG-induced immune response is also The Journal of Immunology 4907 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Conjugation of optimal mouse CpG ODN to CTB augments the immunostimulatory effects of CpG ODN in mouse splenocytes. A–D, Splenocytes from C57BL/6 mice were incubated with different concentrations of CpG 1826, CTB ϩ CpG 1826, or CTB-CpG 1826. The supernatants were analyzed after 24 h for MIP-1␣, MIP-1␤, and IL-6 levels by ELISA, and proliferative response was examined on day 3. E and F, Splenocytes from C57BL/6 mice were incubated with CTB (20 ␮g/ml), maleimide-activated CTB (20 ␮g/ml), CpG 1826 (3.3 ␮g/ml), or thiolated CpG 1826 (3.3 ␮g/ml). The levels of MIP-1␣ in the supernatants (24 h) and cell proliferative responses (day 3) were examined. Data shown are representative of ﬁve independent experiments and expressed as mean of duplicate ELISA samples or triplicate proliferation samples Ϯ SEM.

dependent on endosomal acidification, murine splenocytes were PI3K is involved in CTB-CpG-induced immune activation pretreated with chloroquine or quinacrine, known as blockers of PI3K is an important mediator of TLR9 signaling by CpG (20). To endosomal acidification (18, 19), followed by an overnight incu- examine the involvement of PI3K in CTB-CpG-induced immune bation with CpG ODN or CTB-CpG. As depicted in Fig. 6A, chlo- responses, mouse splenocytes were incubated with wortmannin roquine and quinacrine reduced the CpG-induced MIP-1␣ produc- (WMN), a PI3K inhibitor (20), for 2 h followed by overnight in- tion by 11- and 21-fold, respectively. CTB-CpG-induced MIP-1␣ cubation of the cells with CpG or CTB-CpG. As shown in Fig. 6B, production, in contrast, was not reduced by chloroquine or quin- WMN completely blocked the CpG-induced MIP-1␣ production. acrine. Chloroquine and quinacrine reduced the CpG-induced WMN reduced the CTB-CpG-induced MIP-1␣ by 2-fold. Similar MIP-1␤ production by 7- and 11-fold, respectively. However, pattern was observed in MIP-1␤ production where CTB-CpG re- these inhibitors induced only a marginal reduction in CTB-CpG- ␤ induced MIP-1␤ response (data not shown). Similar to CpG, duced MIP-1 production by 3-fold when cells had been pre- MIP-1␣ and MIP-1␤ productions induced by CTB ϩ CpG were treated with WMN (data not shown). Thus, it appears that PI3K dramatically reduced by pretreatment of the cells with chloroquine activity is involved in CTB-CpG-induced immunity. or quinacrine (data not shown). Consistent with the chemokine data, the CpG ODN-induced proliferative response was markedly Inhibitory ODN 1502 singly or in conjugation with CTB fails to reduced by chloroquine and quinacrine (18-fold reduction), inhibit the CTB-CpG-induced chemokine response whereas these inhibitors did not affect the CTB-CpG-induced proliferative response (data not shown). Together, these results Inhibitory ODNs that contain poly G or GC sequences have been indicate that blocking of endosomal acidification, which dramati- shown to specifically inhibit CpG-induced immunity. Therefore, cally inhibits the CpG-induced immune response, does not abolish we wanted to evaluate the effect of inhibitory ODN 1502, with CTB-CpG-induced immune responses. known inhibitory effect on CpG (21) on induction of immunity by 4908 NOVEL IMMUNOSTIMULATORY AGENT BASED ON CpG DNA LINKED TO CTB

FIGURE 4. CpG sequence is important for the enhanced immunostimulatory effects of CTB-CpG in mouse splenocytes. Splenocytes from C57BL/6 mice were incubated with CpG 1826, CTB-CpG 1826, GpC ODN, or CTB-GpC. The supernatants were analyzed after 24 h for MIP-1␣ (A) and MIP-1␤ (B). Proliferative response (C) was examined after 72 h. Results shown are representative of two independent experiments and expressed as mean of duplicate ELISA samples or triplicate proliferation samples Ϯ SEM.

CTB-CpG. Murine splenocytes were cultivated with CpG or CTB- imply that the inhibitory ODN singly or in conjugation with CTB CpG in the presence or absence of increasing concentrations of fails to inhibit the CTB-CpG-induced chemokine response. inhibitory ODN. Inhibitory ODN dose-dependently suppressed the

CpG-induced MIP-1␣ production. Thus, a concentration of 9 CTB-CpG-induced immune response is dependent on TLR9 and Downloaded from ␮g/ml inhibitory ODN completely blocked MIP-1␣ production MyD88 (Fig. 6C) and induced a 5-fold reduction in MIP-␤ response (data To investigate whether TLR9 and MyD88 are involved in CTB- not shown). CTB-CpG-induced MIP-1␣ (Fig. 6D) and MIP-1␤ CpG signaling, splenocytes from C57BL/6, TLR9Ϫ/Ϫ, and responses (data not shown), in contrast, were not affected by the MyD88Ϫ/Ϫ mice were cultivated in the presence of CpG ODN or inhibitory ODN at any concentration used. CTB-CpG. Splenocytes from C57BL/6 mice produced a signiﬁ-

Next, we addressed the question of whether inhibitory ODN cant amount of MIP-1␣ in response to CpG ODN, which was http://www.jimmunol.org/ linked to CTB (CTB-inhib) can inhibit the immunostimulatory ef- further enhanced by 5-fold in response to CTB-CpG (data not fects of CTB-CpG. To this end, murine splenocytes were incu- shown). As expected, splenocytes from TLR9Ϫ/Ϫ and MyD88 Ϫ/Ϫ bated with increasing concentrations of CTB-inhib conjugate. As mice had no MIP-1␣ response to CpG ODN. Similarly, CTB-CpG depicted in Fig. 6E, CTB-inhib conjugate did not significantly in- did not induce any MIP-1␣ response in splenocytes from TLR9Ϫ/Ϫ hibit the CTB-CpG induced MIP-1␣ response; however, a slight or MyD88Ϫ/Ϫ mice. However, splenocytes from TLR9Ϫ/Ϫ mice reduction in MIP-1␣ response was observed when a concentration produced significant amounts of MIP-1␣ in response to LPS while of 9 ␮g/ml CTB-inhib was used. Taken together, these results no detectable level of MIP-1␣ was produced by splenocytes from MyD88Ϫ/Ϫ mice cultivated with LPS (Fig. 7A). Similar to C57BL/6 mice, splenocytes from TLR9ϩ/Ϫ heterozygote mice by guest on September 28, 2021 produced elevated levels of MIP-1␣ in response to CpG and CTB- CpG (data not shown). Neither CpG ODN nor CTB-CpG induced any appreciable levels of proliferative responses in splenocytes from TLR9Ϫ/Ϫ and MYD88Ϫ/Ϫ mice. However, LPS induced a similar significant level of proliferative response in splenocytes from C57BL/6 and TLR9Ϫ/Ϫ mice (Fig. 7B). These data indicate that similar to CpG, the CTB-CpG-induced immune response is critically dependent on TLR9 and MyD88.

Enhanced immune response elicited by CTB-CpG is not attributed to LPS and is not mediated by TLR4 We evaluated the possible involvement of LPS and also TLR4 in CTB-CpG-induced immune responses by using C3H/HeJ (TLR4- deficient) and C3H/HeN (wild-type) mice. Thus, splenocytes from C3H/HeN and C3H/HeJ mice were cultivated in the presence of CpG or CTB-CpG. CpG induced similar levels of MIP-1␣, MIP- 1␤, and proliferative responses in splenocytes from C3H/HeN and C3H/HeJ mice (Fig. 7, A and B, and data not shown). Likewise, comparable levels of MIP-1␣, MIP-1␤, and proliferative responses were mounted by splenocytes from C3H/HeN and C3H/HeJ mice FIGURE 5. Incubation with GM1 ganglioside diminishes the immuno- in response to CTB-CpG. As expected, splenocytes from C3H/ stimulatory effects of CTB-CpG. A and B, CpG 1826 (0.3 ␮g/ml) or CTB- HeN, but not C3H/HeJ, mice proliferated and produced significant CpG 1826 (CpG content: 0.3 ␮g/ml) were preincubated at 37°C for 30 min amounts of MIP-1␣ and MIP-1␤ in response to LPS (Fig. 7, A and ␮ in the presence or absence of GM1 ganglioside (2.5 or 10 g/ml). Spleno- B, and data not shown). We further investigated the possible in- cytes from C57BL/6 mice were incubated with the preincubated reagents. ␣ volvement of LPS in CTB-CpG-induced immune responses by in- MIP-1 was measured in the supernatants by ELISA after overnight in- ϩ cubation (A). Cell proliferative responses were examined on day 3 (B). cubation of splenocytes from C57BL/6 mice with CpG, CTB Results shown are representative of two different experiments and are pre- CpG, or CTB-CpG in the presence or absence of polymyxin B. As sented as the mean of duplicate ELISA samples or triplicate proliferation shown in Fig. 7C, comparable levels of MIP-1␣ were produced by Statistically significant by Student’s t test at the splenocytes in the presence or absence of polymyxin B. These ,ءء and ء .samples Ϯ SEM p Ͻ 0.05 and p Ͻ 0.01, respectively, compared with groups without GM1. data clearly demonstrate that the enhanced immunostimulatory The Journal of Immunology 4909 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. CTB-CpG-induced immune responses are independent of endosomal acidification and resistant to inhibitory ODN but dependent of PI3K activity. A and B, Murine C57BL/6 splenocytes were preincubated with chloroquine (4 ␮g/ml), quinacrine (0.4 ␮g/ml) (A), or wortmannin (WMN) (2.5 ␮g/ml) (B)for2hat37°C, followed by cultivation with CpG 1826 (0.3 ␮g/ml) or CTB-CpG 1826 (CpG content: 0.3 ␮g/ml). C–E, Splenocytes from C57BL/6 mice were incubated with different concentrations of inhibitory ODN 1502 or CTB-inhibitory ODN 1502 together with CpG (0.3 ␮g/ml) or CTB-CpG (CpG content: 0.3 ␮g/ml). MIP-1␣ response after overnight incubation was examined by ELISA. The figures show one representative experiment .Statistically significant by Student’s t test at p Ͻ 0.01 compared with groups without inhibitors ,ءء .(of two or three (mean Ϯ SEM

property of CTB-CpG is not mediated by LPS and that TLR4 is not absence of gliotoxin for 2 h. The preincubated cells were then involved in CTB-CpG-induced immune responses. exposed to CpG ODN 1826 or CTB-CpG 1826 for 4 and 24 h. The treatment of the cells with gliotoxin abolished the MIP-1␣ and ␬ CTB-CpG-induced immune response is dependent on NF- B proliferative responses produced by CpG and CTB-CpG at both Many genes involved in immunostimulatory responses are acti- time points (data not shown). Hence, similar to CpG, the CTB- vated by the transcription factor NF-␬B. To investigate whether CpG-induced immune response is mediated by NF-␬B. CTB-CpG activation of TLR9-MyD88 signaling was mediated through NF-␬B, human PBMCs were exposed to CTB, CpG 2006, Conjugation of CpG to CTB confers an enhanced cross-species CTB ϩ CpG 2006, or CTB-CpG 2006 for up to 2 h. Nuclear activity to CpG extracts were prepared and subjected to EMSA and supershift Different CpG motifs are optimal to activate murine and human analysis for determination of NF-␬B DNA-binding activity. Nu- immune cells. Thus, PO ODNs containing the optimal murine CpG clear extracts from nonstimulated cells were used as controls. As motif, used at concentrations that are highly active on murine im- illustrated in Fig. 8, NF-␬B was activated by CTB-CpG conjugate mune cells, showed little or no immunostimulatory activity on hu- and to a lesser extent by CpG ODN, with p65 identified as the man immune cells (22). We were interested to know whether con- major activated NF-␬B subunit. Minimal activation of NF-␬B jugation of CTB to CpG can override the species specificity effect above that observed with control cells occurred when CTB and of CpG. Hence, human PBMCs were cultivated in the presence of CTB ϩ CpG ODN were tested. To further confirm the involve- increasing concentrations of optimal mouse CpG 1826, CTB ϩ ment of NF-␬B, the impact of gliotoxin, a specific inhibitor of CpG 1826, or CTB conjugated to CpG 1826. Incubation of human NF-␬B on the CTB-CpG-induced immune responses were inves- PBMCs with optimal mouse CpG 1826 at a concentration of 27 tigated. Mouse splenocytes were preincubated in the presence or ␮g/ml induced a 2-fold increase in MIP-1␣ production, which was 4910 NOVEL IMMUNOSTIMULATORY AGENT BASED ON CpG DNA LINKED TO CTB

FIGURE 8. Conjugation of CTB to CpG ODN augments NF-␬B activation. Human PBMCs were either unstimulated (1) or stimulated with CTB (34.9 ␮g/ml) (2), CpG 2006 (9 ␮g/ml) (3), CTB (CTB: 34.9 ␮g/ml) ϩ CpG 2006 (9 ␮g/ml) (4), or human CTB-CpG 2006 (CpG content: 9 ␮g/ml) (5). Nuclear extracts were prepared from cells following incubation periods 30 min to 2 h. NF-␬B-binding activity was determined by EMSA (5% gels using an oligonucleotide probe containing the consensus NF-␬B sequence). Identification of NF-␬B Rel A subunit in the DNA-protein complexes was determined in the presence (shifted) and absence (unshifted) of specific Rel A Ab (1 ␮g/sample). Two hour data is illustrated. The figure shows one representative experiment. Downloaded from Next, we examined whether the cross-species activity of CpG ODN was mediated by TLR9 and MyD88. Thus, splenocytes from TLR9Ϫ/Ϫ and MyD88Ϫ/Ϫ mice were cultivated with human CpG 2006, CTB ϩ CpG 2006, or CTB- CpG 2006. As depicted in Fig. 9, C and D, incubation of splenocytes from TLR9Ϫ/Ϫ and Ϫ/Ϫ ϩ MyD88 mice with CpG 2006, CTB CpG 2006, or CTB-CpG http://www.jimmunol.org/ 2006 did not result in any significant MIP-1␣ and MIP-1␤ responses. Together, these results indicate that conjugation of CpG ODN to CTB confers an enhanced cross-species activity to CpG ODN and that the cross-species activity of CTB-CpG is also me- FIGURE 7. TLR9 and MYD88, but not TLR4, are important for the diated by TLR9 and MyD88. CTB-CpG-induced immune responses. A and B, Splenocytes from C3H/ HeN, C3H/HeJ, TLR9Ϫ/Ϫ, and MYD88Ϫ/Ϫ mice were cultured with CpG Conjugation of CTB to CpG enhances adjuvanticity of CpG 1826 (0.3 ␮g/ml) or CTB-CpG (CpG content: 0.3 ␮g/ml). LPS (5 ␮g/ml) Given the potent immunostimulatory effects of CTB-CpG, we was included as a positive control. MIP-1␣ (A) and proliferative (B) re- sought to compare the adjuvanticity of CTB-CpG with CpG, CTB, by guest on September 28, 2021 sponses were examined after overnight and 3 days incubation, respectively. and CTB ϩ CpG. Groups of C57BL/6 mice were s.c. injected with ␮ C, Splenocytes from C57BL/6 were incubated with CpG 1826 (0.3 g/ml), 10 ␮g of TT alone, or together with either 5 ␮g of CpG ODN, 18 CTB ϩ CpG or CTB-CpG (CpG content: 0.3 ␮g/ml) in the presence or ␮g of CTB, or equivalent amounts of CpG or CTB in the mixture absence of polymyxin B (PmB) (10 ␮g/ml) overnight. Results are representative of three different experiments (mean Ϯ SEM). or conjugate, two times, with a 1-wk interval. Three weeks after the last immunization, the level of serum IgG Ab against TT was examined. The TT vaccinated group had considerable amounts of not further enhanced when CTB ϩ CpG was used. CTB-CpG TT-specific IgG in their sera. Immunization with TT together with 1826, in contrast, induced a robust MIP-1␣ response in a dose- CTB, CpG ODN, or CTB ϩ CpG induced almost 2-fold increase dependent manner. Thus, a concentration of 27 ␮g/ml CTB-CpG in TT-specific IgG Ab responses compared with TT immunization induced a 418-fold increase in MIP-1␣ production over untreated alone. However, mice immunized with TT together with CTB- PBMCs (Fig. 9A). CpG (27 ␮g/ml) induced a 19-fold increase in CpG induced a 6-fold increase in their TT-specific IgG Abs as MIP-1␤ response compared with untreated PBMCs that was not compared with TT group (Fig. 10A). These data imply that CTB- further increased when CTB ϩ CpG was used. Conversely, incu- CpG is a strong adjuvant for induction of systemic Ab responses bation of human PBMCs with CTB-CpG resulted in a 205-fold against a coadministered Ag. increase in MIP-1␤ level as compared with untreated PBMCs (Fig. Next, we tested the levels of IgG1 and IgG2c Abs as indicator 9B). Thus, conjugation of optimal murine CpG to CTB confers a of Th2 and Th1 type responses in C57BL/6 mice, respectively, in strong immunostimulatory effect on human PBMCs. the sera of immunized mice. As depicted in Fig. 10B, all vacci- We then tested the immunostimulatory effects of human CpG nated groups had comparable levels of a specific IgG1 Ab re- 2006 linked to CTB in murine splenocytes. Splenocytes from sponse. By contrast, the level of IgG2c Ab varied. Thus, mice C57BL/6 mice produced moderate levels of MIP-1␣ (4000 pg/ml/ immunized with TT, or TT coadministered with CTB or CTB ϩ million cells) and MIP-1␤ (10 000 pg/ml/million cells) in response CpG, had low levels of serum IgG2c. Mice immunized with TT to CpG 2006 (3 ␮g/ml) that was not further enhanced when CTB together with CpG had moderate levels of IgG2c (a 2-fold increase ϩ CpG 2006 was used. CTB-CpG 2006 induced a 3-fold increase over TT alone), whereas immunization with TT coadministered in MIP-1␣ and MIP-1␤ levels over those of CpG 2006 (Fig. 9, C with CTB-CpG induced a dramatic increase in the TT-specific and D). We also tested the requirement of CpG sequence in the IgG2c levels (a 6-fold increase over TT alone), indicating of a cross-species activity of CTB-CpG in murine splenocytes. Incu- strong Th1-tilting effect of CTB-CpG on immune response in vivo bation of splenocytes from C57BL/6 mice with non-CpG-contain- (Fig. 10C). ing control ODN IMTO22 induced virtually no MIP-1␣, MIP-1␤, We then examined IFN-␥ response, a bona fide marker of cel- and IL-6 responses. However, CTB-IMTO22 induced a slight in- lular immune response, in splenocytes isolated from the immu- crease in cytokine/chemokine responses (data not shown). nized mice upon Ag recall stimulation in vitro. Splenocytes from The Journal of Immunology 4911 Downloaded from

FIGURE 9. A B Conjugation of CTB to CpG overrides species-specificity property of CpG. and , Human PBMCs were cultivated with different http://www.jimmunol.org/ concentrations of mouse CpG 1826 (mCpG), CTB ϩ mCpG or mCTB-CpG. The supernatants were analyzed for MIP-1␣ and MIP-1␤ at 24 h. C and D, Splenocytes from C57BL/6, TLR9Ϫ/Ϫ, and MYD88Ϫ/Ϫ mice were incubated with human CpG 2006 (3 ␮g/ml) (hCpG), CTB (11.6 ␮g/ml) ϩ hCpG or hCTB-CpG (CTB: 11.6 ␮g/ml and CpG: 3 ␮g/ml). The supernatants were analyzed for MIP-1␣ and MIP-1␤ at 24 h. The figures show one representative experiment of two (mean Ϯ SEM). the immunized mice were cultured with TT for 48 h and IFN-␥ timal human CpG 2006 linked to CTB was shown to mount robust production was analyzed in cell culture supernatants. Although proinflammatory cytokine and chemokine responses in human splenocytes from mice immunized with TT alone or TT together PBMCs while CpG 2006 alone or an admixture of CTB and CpG by guest on September 28, 2021 with CTB, CpG ODN, or CTB ϩ CpG ODN did not produce any 2006 induced only weak responses. Also in mouse splenocytes, appreciable levels of IFN-␥, splenocytes from mice immunized optimal murine CpG linked to CTB mounted a more potent with TT together with CTB-CpG produced considerable amounts proinflammatory cytokine and chemokine responses as well as of IFN-␥ in vitro (Fig. 10D). proliferative response than CpG alone or admixture of CTB and CpG. Interestingly, the well-documented species specificity of Discussion CpG ODN appears to be largely overridden by the binding to CTB. In this study, we describe the development and properties of a Of special significance, CTB-CpG displayed a markedly aug- potent novel immunostimulatory agent based on chemical conju- mented in vivo adjuvant activity for a coadministered vaccine Ag, gation of CpG ODN to CTB. The rationale for this construction TT compared with CpG alone, or a mixture of CTB and CpG. We involved the possibility of combining the immunostimulatory also report a series of studies that analyze mode of action of properties of CpG ODN with the efficient ability of CTB to bind to CTB-CpG. In the following, we attempt to discuss several possi- immune cells, especially APCs, and to transport linked agent into bilities that may depict the underlying mechanism for the enhanced endosomes and retrogradely further to the TGN and the ER. Op- immunostimulatory properties of CTB-CpG.

FIGURE 10. Systemic IgG Ab responses in mice immunized with TT and different adjuvants. Groups of mice were immunized s.c. with TT (10 ␮g) alone or in combination with CTB (18 ␮g), CpG (5 ␮g), CTB (18 ␮g) ϩ CpG (5 ␮g), or CTB-CpG (CTB: 18 ␮g, CpG: 5 ␮g) three times 7–10 days apart. Two weeks after the last immunization serum, IgG (A) and IgG isotypes (B and C) as well as IFN-␥ response in in vitro splenocytes culture (D) were examined. IgG Ab responses are expressed as reciprocal IgG titer and OD value of 1/500 prediluted sera measured at 450 nm. Data shown are representative of three experiments Statistically signiﬁcant ,ءء .(mean Ϯ SEM of n ϭ 3) by Student’s t test at p Ͻ 0.01 compared with TT group (IgG2c) or control group (IFN-␥). 4912 NOVEL IMMUNOSTIMULATORY AGENT BASED ON CpG DNA LINKED TO CTB

We could show that the immunostimulatory properties of CTB- Another interesting feature of CTB-CpG explained in our study CpG are dependent on the presence of CpG motif as a non-CpG- is its potent adjuvant activity for induction of an Ag-specific IgG containing ODN conjugated to CTB failed to elicit robust proin- Ab response when used for s.c. immunization together with a flammatory cytokine and chemokine responses in human PBMCs. model vaccine Ag, TT, in C57BL/6 mice. We found that CTB- Furthermore, and despite dissimilarities between CpG and CTB- CpG is especially capable of mobilizing more potent IgG2c Ab CpG discussed below, several lines of evidence in the present and IFN-␥ responses than CpG, indicating that coupling of CpG to study suggest that CTB-CpG shares critical molecules involved in CTB tilts the resulting immune response toward Th1 type immu- TLR9 signaling with CpG. Thus, similar to CpG, the CTB-CpG- nity. Previous studies point to a synergistic adjuvant activity induced immune response was found to be dependent on TLR9, between CpG ODN and CT or LT for induction of Ag-specific MyD88, PI3K, and NF-␬B. immune responses in mice. In one study, transcutaneous immuni- In our mouse in vitro experiments, we could show that the CpG zation of mice with Chlamydia trachomatis major outer membrane dose required for eliciting proinflammatory cytokine and chemo- protein as Ag and a mixture of CpG and CT as adjuvant was shown kine responses as well as proliferative response can be dramati- to be superior to CpG alone in inducing immune responses against cally reduced if CpG is linked to CTB. Furthermore, preincubation the coadministered Ag (10). However, the inherent toxicity of CT of CTB-CpG with ganglioside GM1 was shown to decrease the and LT precludes their use as adjuvants in humans. Consistent with response produced by CTB-CpG to that of CpG ODN. It is there- our findings, a mixture of CpG ODN and nontoxic derivatives of fore likely that, in addition to the endosomal formation evidenced CT and LT, i.e., CTB and the mutant of LT protein derivative for CpG uptake, the cellular uptake of CTB-CpG through binding LTK63, had no synergistic effects on the magnitude of the Ag- to the GM1 receptor allows more CpG to reach the endosomal specific immune response (9). Conjugation of CpG and CTB may Downloaded from compartments. Of note, the recent report of Yasuda et al. (23) be a means of overcoming these functional limitations. revealed that enhancement of endosomal translocation of CpG In conclusion, we described here the development and proper- DNA by using the cationic lipid DOTAP augments the immuno- ties of a potent immunostimulatory agent based on chemical con- stimulatory effects of CpG DNA in dendritic cells in terms of up- jugation of CpG ODN to CTB. We found that the immunostimu- regulation of CD40/CD69 and production of type I IFN and IL-6. latory and the adjuvant effects of CpG can be dramatically

Recent studies of Leifer et al. (24) demonstrated that TLR9 is enhanced by the conjugation to CTB. To our knowledge, we are http://www.jimmunol.org/ localized in the ER before CpG exposure. Concurrent with the the first to report CTB as the first nontoxic derivative of microbial movement of CpG in target cells, TLR9 redistributes from the ER toxins discovered that when chemically linked to CpG ODN dra- to the endosome (25). Internalized CTB has been found to traffic matically enhances the CpG ODN-mediated immune responses. intracellularly via a retrograde vesicular pathway to reach the This observation warrants further exploration of the impact of the TGN, Golgi apparatus, and eventually the ER (13). One possibility new class of immunostimulator/adjuvant based on CpG ODN is that CTB-CpG might follow the route used by CTB and thereby linked to CTB for inducing more potent immune responses. translocates to the ER where CpG can primarily interact with TLR9. CpG-TLR9 may then translocate from the ER to the endo- Acknowledgments somal vesicles where the TLR9 signaling takes place. It has been We gratefully acknowledge Prof. Mary Jo Wick (Go¨teborg University, by guest on September 28, 2021 recently reported that retention of CpG in the endosomal vesicle Go¨teborg, Sweden) for providing TLR9Ϫ/Ϫ and MyD88Ϫ/Ϫ mice. We are may enhance the avidity of CpG to TLR9. Thus, monocyte-derived grateful to Karolina Tho¨rn and Elizabeth Logan for skilled technical human DCs that respond poorly to CpG-A mount a robust IFN assistance. induction if CpG-A is manipulated using a cationic lipid for endosomal retention (26). It is therefore possible that conjugated Disclosures CTB-CpG is retained for longer periods in the endosomal vesicle The authors have no financial conflict of interest. thus enhancing the avidity of CpG to TLR9. Cell imaging studies are underway to investigate this possibility. Additionally, the re- References sistance of the immune responses produced by CTB-CpG to the 1. Weiner, G. J. 2000. The immunobiology and clinical potential of immunostimulatory CpG oligodeoxynucleotides. J. Leukocyte Biol. 68: 455–463. inhibitory ODN suggests that unlike CpG, the interaction of CTB- 2. Krieg, A. M. 2002. CpG motifs in bacterial DNA and their immune effects. Annu. CpG with TLR9 does not primarily take place in endosomal ves- Rev. Immunol. 20: 709–760. icles and further stress our notion that the ER might be involved in 3. Harandi, A. M., and J. Holmgren. 2004. CpG DNA as potent inducer of mucosal immunity: implications for immunoprophylaxis and immunotherapy of mucosal this process. Nonetheless, the inhibitory ODN linked to CTB failed infections. Curr. Opin. Invest. Drugs 5: 141–145. to diminish the CTB-CpG-induced immune response. This illus- 4. Pisetsky, D. S., and C. Reich. 1993. Stimulation of in vitro proliferation of murine trates the complexity of the underlying mechanism of CTB-CpG. lymphocytes by synthetic oligodeoxynucleotides. Mol. Biol. Rep. 18: 217–221. 5. Hacker, G., V. Redecke, and H. Hacker. 2002. Activation of the immune system Recent observations by Wu et al. (27) indicate that multimer- by bacterial CpG-DNA. Immunology 105: 245–251. ization of CpG ODN plays a pivotal role for TLR9 activation and 6. Klinman, D. M., A. K. Yi, S. L. Beaucage, J. Conover, and A. M. Krieg. 1996. CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete in- that CpG PO can also activate TLR9 if they form a multimer terleukin 6, interleukin 12, and interferon ␥. Proc. Natl. Acad. Sci. USA 93: structure. Our findings show that CpG PO linked to CTB can 2879–2883. mount a potent immune response. By analogy, it is therefore pos- 7. Hartmann, G., and A. M. Krieg. 2000. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J. Immunol. 164: sible that coupling of CTB, which has a pentameric structure, to 1617–1624. CpG effectively, confers CpG aggregation/multimerization that 8. Fearon, K., J. D. Marshall, C. Abbate, S. Subramanian, P. Yee, J. Gregorio, may subsequently lead to a more effective interaction of CpG with G. Teshima, G. Ott, S. Tuck, G. Van Nest, and R. L. Coffman. 2003. A minimal human immunostimulatory CpG motif that potently induces IFN- and IFN- pro- TLR9. Consistent with this notion, it has been recently shown that duction. Eur. J. Immunol. 33: 2114–2122. adsorption of CpG ODN to the surface of cationic poly (D,L-lac- 9. McCluskie, M. J., R. D. Weeratna, and H. L. Davis. 2000. Intranasal immuni- ␥ zation of mice with CpG DNA induces strong systemic and mucosal responses tide-co-glycolide) microspheres enhances the secretion of IFN- that are influenced by other mucosal adjuvants and antigen distribution. Mol. and IFN-␣ from human PBMC compared with CpG ODN alone Med. 6: 867–877. (8). Several studies have also shown that CpG ODN linked to 10. Berry, L. J., D. K. Hickey, K. A. Skelding, S. Bao, A. M. Rendina, P. M. Hansbro, C. M. Gockel, and K. W. Beagley. 2004. Transcutaneous immu- protein Ags elicit a more potent Ag-specific response than admix- nization with combined cholera toxin and CpG adjuvant protects against Chla- ture of CpG ODN and Ag (28–30). mydia muridarum genital tract infection. Infect. Immun. 72: 1019–1028. The Journal of Immunology 4913

11. McCluskie, M., and H. L. Davis. 1998. CpG DNA is a potent enhancer of sys- 22. Hartmann, G., R. Weeratna, Z. Ballas, P. Payette, S. Blackwell, I. Suparto, temic and mucosal immune responses against hepatitis B surface antigen with W. Rasmussen, M. Waldschmidt, D. Sajuthi, R. Purcell, et al. 2000. Delineation intranasal administration to mice. J. Immunol. 161: 4463–4466. of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune 12. Holmgren, J. 1981. Actions of cholera toxin and the prevention and treatment of responses in vitro and in vivo. J. Immunol. 164: 1617–1624. cholera. Nature 292: 413–417. 23. Yasuda, K., P. Yu, C. J. Kirschning, B. Schlatter, F. Schmitz, A. Heit, S. Bauer, 13. de Haan, L., and T. R. Hirst. 2004. Cholera toxin: a paradigm for multi-functional H. Hochrein, and H. Wagner. 2005. Endosomal translocation of vertebrate DNA engagement of cellular mechanisms. Mol. Membrane Biol. 21: 77–92. activates dendritic cells via TLR9-dependent and -independent pathways. J. Im- 14. Holmgren, J., A. M. Harandi, and C. Czerkinsky. 2003. Mucosal adjuvants and munol. 164: 1617–1624. anti-infection and anti-immunopathology vaccines based on cholera toxin, chol- 24. Leifer, C. A., M. N. Kennedy, A. Mazzoni, C. Lee, M. J. Kruhlak, and era toxin B subunit and CpG DNA. Expert Rev. Vaccines 2: 205–217. D. M. Segal. 2004. TLR9 is localized in the endoplasmic reticulum prior to 15. Back, E., A. M. Svennerholm, J. Holmgren, and R. Mollby. 1979. Evaluation of stimulation. J. Immunol. 173: 1179–1183. a ganglioside immunosorbent assay for detection of Escherichia coli heat-labile 25. Latz, E., A. Schoenemeyer, A. Visintin, K. A. Fitzgerald, B. G. Monks, enterotoxin. J. Clin. Microbiol. 10: 791–795. C. F. Knetter, E. Lien, N. J. Nilsen, T. Espevik, and D. T. Golenbock. 2004. 16. Schnare, M., A. C. Holt, K. Takeda, S. Akira, and R. Medzhitov. 2000. Recog- TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. nition of CpG DNA is mediated by signaling pathways dependent on the adaptor Immunol. 5: 190–198. protein MyD88. Curr. Biol. 10: 1139–1142. 26. Honda, K., Y. Ohba, H. Yanai, H. Negishi, T. Mizutani, A. Takaoka, C. Taya, and 17. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, T. Taniguchi. 2005. Spatiotemporal regulation of MyD88-IRF-7 signalling for K. Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A Toll-like receptor robust type-I interferon induction. Nature 434: 1035–1040. recognizes bacterial DNA. Nature 408: 740–745. 27. Wu, C. C., J. Lee, E. Raz, M. Corr, and D. A. Carson. 2004. Necessity of oli- 18. Manzel, L., L. Strekowsk, F. M. Ismail, J. C. Smith, and D. E. Macfarlane. 1999. gonucleotide aggregation for Toll-like receptor 9 activation. J. Biol. Chem. 279: Antagonism of immunostimulatory CpG-oligodeoxynucleotides by 4-amin- 33071–33078. oquinolines and other weak bases: mechanistic studies. J. Pharmacol. Exp. Ther. 28. Maurer, T., A. Heit, H. Hochrein, F. Ampenberger, M. O’Keeffe, S. Bauer, 291: 1337–1347. G. B. Lipford, R. M. Vabulas, and H. Wagner. 2002. CpG-DNA aided cross- 19. Macfarlane, D. E., and L. Manzel. 1998. Antagonism of immunostimulatory presentation of soluble antigens by dendritic cells. Eur. J. Immunol. 32: CpG-oligodeoxynucleotides by quinacrine, chloroquine, and structurally related 2356–2364. compounds. J. Immunol. 160: 1122–1131. 29. Heit, A., F. Schmitz, M. O’Keeffe, C. Staib, D. H. Busch, H. Wagner, and Downloaded from 20. Ishii, K. J., F. Takeshita, I. Gursel, M. Gursel, J. Conover, A. Nussenzweig, and K. M. Huster. 2005. Protective CD8 T cell immunity triggered by CpG-protein D. M. Klinman. 2002. Potential role of phosphatidylinositol 3 kinase, rather than conjugates competes with the efﬁcacy of live vaccines. J. Immunol. 174: DNA-dependent protein kinase, in CpG DNA-induced immune activation. J. Exp. 4373–4380. Med. 196: 269–274. 30. Shirota, H., K. Sano, N. Hirasawa, T. Terui, K. Ohuchi, T. Hattori, K. Shirato, 21. Yamada, H., I. Gursel, F. Takeshita, J. Conover, K. J. Ishii, M. Gursel, and G. Tamura. 2001. Novel roles of CpG oligodeoxynucleotides as a leader for S. Takeshita, and D. M. Klinman. 2002. Effect of suppressive DNA on CpG- the sampling and presentation of CpG-tagged antigen by dendritic cells. J. Im- induced immune activation. J. Immunol. 169: 5590–5594. munol. 167: 66–74. http://www.jimmunol.org/ by guest on September 28, 2021