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Originally published online as doi:10.1189/jlb.0407236 on October 29, 2007

Published online before print October 29, 2007
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(Journal of Leukocyte Biology. 2008;83:381-392.)
© 2008 by Society for Leukocyte Biology

{alpha}2-Macroglobulin binds CpG oligodeoxynucleotides and enhances their immunostimulatory properties by a receptor-dependent mechanism

Ryan B. Anderson, George J. Cianciolo, Margaret N. Kennedy and Salvatore V. Pizzo1

Department of Pathology, Duke University Medical Center, Durham, North Carolina, USA

1 Correspondence: Department of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710, USA. E-mail: pizzo001{at}mc.duke.edu


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ABSTRACT
 
CpG oligodeoxynucleotides (ODN) stimulate the immune system and are under evaluation as treatments and vaccine adjuvants for infectious diseases, cancer, and immune system disorders. Although they have shown promising results in numerous clinical trials, the ultimate use of CpG ODN-based therapeutics may hinge on improved pharmacokinetics and reduced systemic side-effects. CpG ODN efficacy and potency might be enhanced greatly by packaging them into particles that protect them from degradation and specifically target them for uptake by immune-competent cells. The plasma proteinase inhibitor {alpha}2-macroglobulin ({alpha}2M) binds numerous biologically active macromolecules, including cytokines, chemokines, and growth factors, and can modulate their activity. Molecules bound to {alpha}2M are protected from interactions with neighboring macromolecules and are targeted for receptor-mediated uptake by immune-competent cells. Here, we report that activated {alpha}2M ({alpha}2M*) binds CpG ODN and enhances their immunostimulatory properties significantly. Murine macrophages treated with {alpha}2M*-ODN complexes respond more rapidly and produce a greater cytokine response than induced by free CpG ODN. Using human PBMC, {alpha}2M*-ODN complexes exhibit fourfold enhanced potency and 15-fold greater efficacy for stimulating production of inflammatory cytokines. {alpha}2M* targets delivery of CpG ODN specifically to immune-competent cells, which endocytose the complexes sixfold more rapidly than free CpG ODN. CpG ODN bound to {alpha}2M* are also protected from degradation by nucleases. This novel targeting technology may improve CpG ODN-based therapeutics by increasing efficacy at reduced doses, thus reducing side-effects and cost.

Key Words: vaccine adjuvant • TLR9 • dendritic cells • LRP


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INTRODUCTION
 
During the course of infection, immune-competent cells recognize microbial debris as foreign through a family of TLRs, of which, 10 have been identified in humans. TLRs recognize pathogen-associated molecular patterns, molecular structures widely expressed by pathogens but not by endogenous cells. Bacterial DNA contains unmethylated CpG dinucleotides [1 , 2 ], which are recognized by TLR9 in the endosome [3 4 5 ]. CpG-TLR9 ligation initiates a signaling cascade that activates NF-{kappa}B and induces an inflammatory response [6 ]. The CpG dinucleotide is under-represented significantly in the mammalian genome and is usually methylated when it does occur. Bacterial genomes, however, contain this dinucleotide with the predicted 1:16 frequency [7 ], making it a distinguishing feature between foreign and endogenous DNA. Bacterial DNA and its synthetic mimic CpG oligodeoxynucleotides (CpG ODN) stimulate B cells, monocytes, macrophages (M{Phi}s), plasmacytoid dendritic cells (pDCs), and NK cells. Immune cell activation is marked by up-regulation of MHC classes and costimulatory molecules, and the production of numerous proinflammatory cytokines, such as IL-1, IL-6, IL-12, IFN-{alpha}, IFN-{gamma}, and TNF, which induce a TH1-biased immune response [8 , 9 ]. In vivo, CpG ODN show promise as monotherapies [10 11 12 ] and vaccine adjuvants [13 14 15 ] in the treatment and prevention of infectious diseases. CpG ODN-mediated immune system activation also promotes cancer immunosurveillance, which can slow tumor growth and eradicate some established tumors [16 , 17 ]. There are several clinical trials currently evaluating these applications of CpG ODN [18 ].

Despite numerous promising results, studies also demonstrate potential obstacles to their clinical use. CpG ODN, containing the naturally occurring phosphodiester (P=O) backbone, are degraded rapidly in vivo by nucleases, which prevent the desired immune response [19 ]. Efficacy can be improved with higher doses and repeated dosing; however, this can cause systemic side-effects, including splenomegaly and lethal toxic shock [20 ]. To increase nuclease resistance, the CpG ODN backbone can be modified with phosphorothioate (P=S) linkages, but P = S CpG ODN suffer from their own drawbacks. In addition to side-effects such as granuloma formation [21 ], splenomegaly [22 ], and lymphadenopathy [23 ], they induce an intrinsically different immune response [21 , 24 , 25 ] with inhibition of certain functions of the immune system [26 ]. P = S CpG ODN also bind nonspecifically to plasma proteins, reducing their activity [27 ], and they interact nonspecifically with cell surface receptors, which can disrupt signaling events crucial to normal cellular functions [28 , 29 ]. These shortcomings may be overcome by a molecular delivery mechanism that targets CpG ODN specifically to immune-competent cells and protects them from nonspecific interactions with circulating proteins and cell surfaces.

{alpha}2-Macroglobulin ({alpha}2M) is a plasma proteinase inhibitor that also binds and modulates the activity of numerous effector proteins, including cytokines, chemokines, and growth factors [30 ]. These molecules diffuse into either of two binding pockets of {alpha}2M and are trapped there when {alpha}2M is proteolytically activated; such activation entails a major conformational change that also exposes the receptor-binding sites on each of its four subunits [31 ] (Fig. 1A ). {alpha}2M* [32 ] is internalized, along with the molecules trapped inside, by its endocytic receptor, the low-density lipoprotein receptor-related protein (LRP/CD91), and trafficked to the endosome. LRP/CD91 is a high-affinity receptor [dissociation constant (Kd)=1–5 nM] expressed on numerous cell types, including M{Phi}s, DCs, and B cells [33 , 34 ].


Figure 1
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  		Figure 1. Activated {alpha}2M ({alpha}2M*) binds CpG ODN when converted to its receptor-recognized form. (A) Diagram of {alpha}2M incorporating ligands by proteolytic activation, which leads to a conformational change and conversion to its receptor-recognized form, {alpha}2M*. (B) Coomassie brilliant blue stain of PAGE analysis of {alpha}2M and {alpha}2M* under native conditions. (C) Fluorescently labeled CpG ODN were mixed with {alpha}2M and activated with human neutrophil elastase (HNE); samples were analyzed by PAGE under native conditions. CpG ODN were detected by fluorescent imaging. (D) {alpha}2M* on the same gel was detected by staining with Coomassie brilliant blue. Lanes 1, CpG ODN alone; 2, CpG ODN + {alpha}2M; 3, CpG ODN + {alpha}2M + HNE; 4, {alpha}2M + HNE; 5, {alpha}2M alone.

{alpha}2M* targets its bound molecules to immune-competent cells and enhances their biological activity, a property that can be exploited to modulate immune responses. Mice immunized with a HIV envelope peptide require a 100-fold lower dose of peptide to produce a response when the peptide is incorporated in {alpha}2M* compared with peptide mixed with CFA/IFA or in combination with monophosphoryl lipid/G-CSF [35 ]. {alpha}2M*-antigen complexes of hepatitis B surface antigen also produce antibody responses in mice that are four to five orders of magnitude greater than those elicited by antigen alone [36 ].

We hypothesized that CpG ODN might also incorporate into {alpha}2M*, forming {alpha}2M*-ODN complexes that would serve as a delivery mechanism for therapeutic applications of CpG ODN. {alpha}2M* could serve, not only to protect CpG ODN from nuclease degradation, nonspecific binding to plasma proteins, and renal clearance but also to target CpG ODN to immune-competent cells, thus eliminating nonproductive uptake by irrelevant cells. Such benefits would help to resolve some of the potential barriers already identified for CpG ODN-based therapeutics, including suboptimal efficacy, pharmacokinetics, and systemic side-effects.


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MATERIALS AND METHODS
 
Materials
Fresh frozen human plasma was obtained from the American Red Cross (Durham, NC, USA). CpG ODN were synthesized, labeled, and purified by Midland Certified Reagent Company (Midland, TX, USA); fluorescent label was attached to the 3' end of the CpG ODN, which were then repurified by reversed-phase HPLC; removal of unlabeled CpG ODN and unbound chromophore was verified by mass spectrometry. For murine studies, the CpG ODN sequence used was #1826: 5'-TCCATGACGTTCCTGACGTT-3', which stimulates murine immune cells [37 ]; the control sequence used was identical except that both CpG dinucleotides were inverted to GpC. The sequence used for human PBMC (hPBMC) studies was #2395: 5'-TCGTCGTTTTCGGCGCGCGCCG-3', which induces optimal responses from human cells [38 ], and the control sequence used was similar, except that each cytosine was replaced with guanine, as simple inversion would have yielded new CpG dinucleotides. In all cases, the CpG ODN used were P = S modified, unless otherwise stated. HNE was obtained from Calbiochem (San Diego, CA, USA). Benzonase and DNase I nucleases were obtained from Novagen (Madison, WI, USA). C57BL/6 [wild-type (WT)] female mice, 6–8 weeks old, were obtained from Charles River Laboratories (Raleigh, NC, USA) and fed food and water ad libitum. C57BL/6 female mice deficient in TLR9 [knockout (KO)], 6–8 weeks old, were kindly provided by our collaborator Dr. Greg Sempowski of Duke University Department of Pathology and Duke Vaccine Institute (Durham, NC, USA). Dr. Richard Flavell of Yale University provided mice to Dr. Sempowski. All other reagents were of the highest available grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA).

CpG ODN incorporation into {alpha}2M
{alpha}2M was purified from fresh, frozen plasma, as described previously [32 ]. Labeled ODN #1826 and {alpha}2M were mixed at a molar ratio of 5:1, HNE was added at a 2.5-fold molar excess with respect to {alpha}2M, and the mixture was incubated at 25°C for 30 min to {alpha}2M*. The samples were analyzed by PAGE under native conditions; CpG ODN, bound and free, were detected by fluorescent imaging using a Storm 860 PhosphorImager® (Molecular Dynamics, Sunnyvale, CA, USA) and ImageQuant software. {alpha}2M was detected by staining with Coomassie brilliant blue. Although {alpha}2M and {alpha}2M* cannot be distinguished in Figure 1C , these samples were also analyzed by PAGE under conditions that separate these two conformations of {alpha}2M; they were loaded on 5% Tris-boric acid-EDTA buffer gels (BioRad, Hercules, CA, USA) and electrophoresed at 150 V for 1 h.

To demonstrate binding specificity, labeled CpG ODN were mixed in fivefold molar excess with {alpha}2M, and unlabeled CpG ODN were added in the following molar ratios, with respect to labeled CpG ODN: 0, 1, 2, 5, 10, 25, 50, and 100. HNE was added, and samples were incubated at 25°C for 30 min, followed by PAGE analysis under native conditions. The amounts of labeled CpG ODN that incorporated into {alpha}2M* were quantified by fluorescent imaging as described previously. Gels were stained with Coomassie brilliant blue to verify complete proteolytic conversion of {alpha}2M to {alpha}2M*.

Enhanced biological activity of CpG ODN and {alpha}2M*-ODN
Approval of the following procedure was obtained from Duke University Institutional Animal Care and Use Committee; all experiments were performed in accordance with relevant guidelines and regulations. Thioglycollate (TG)-elicited peritoneal M{Phi}s were harvested from WT or KO C57BL/6 mice by peritoneal lavage and plated in 96-well plates at a concentration of 2.5 x 105 cells per well in DMEM with high glucose, L-glutamine, 110 mg/L sodium pyruvate, pyridoxine hydrochloride, 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin. Cells were incubated at 37°C for 2 h and washed to remove nonadherent cells. Fresh media were added, and cells were incubated at 37°C for 18 h.

{alpha}2M*-ODN complexes were produced by the methods described below; these procedures were used for the remainder of the experiments described in this report. Labeled ODN #1826 and {alpha}2M were mixed in a molar ratio of 10:1, HNE was added, and the mixture was incubated at 25°C for 30 min to activate {alpha}2M. Unbound CpG ODN were separated from {alpha}2M*-ODN complexes using a BioLogic fast protein liquid chromatography (BioRad) with a Superdex 200 10/300 GL size exclusion column (Amersham Biosciences, Piscataway, NJ, USA). Fractions containing complexes were pooled and concentrated using Amicon Centriprep YM-50 filtration units (Millipore, Billerica, MA, USA). Removal of unbound CpG ODN was verified by PAGE separation followed by detection of CpG ODN by fluorescent imaging as described previously. The concentration of CpG ODN in the {alpha}2M*-ODN complexes was measured by fluorescence using a Shimadzu RF-5301 PC spectrofluorophotometer, and {alpha}2M* concentration was measured using the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). LPS contamination of the complexes was undetectable using a kinetic chromogenic limulus amoebocyte lysate assay (kinetic QCL assay; BioWhittaker, Walkersville, MD, USA).

Cells were treated with free or {alpha}2M*-bound CpG ODN at a final CpG ODN concentration ranging from 0.06 to 5.0 µM. Only free CpG ODN was used at 5.0 µM; the solubility of {alpha}2M* prevented the use of {alpha}2M*-ODN complexes at this highest concentration. Cells were incubated for 24 h at 37°C and 5% CO2, after which time, the supernatants were collected. TNF was measured using a DuoSet ELISA kit from R&D Systems (Minneapolis, MN, USA). For the response kinetics studies, the experiment was repeated with the following changes. The concentration of free and {alpha}2M*-bound CpG ODN used was 0.5 µM, and supernatants were collected after 2 h, 4 h, 8 h, 16 h, and 24 h. TNF was measured by ELISA, and a BioPlex kit (BioRad) was used with a Luminex-100 Multiplex instrument to measure IL-1{alpha}, IL-6, IL-12 (p70), and MCP-1. As controls, cells were treated with {alpha}2M* alone and with a control sequence ODN bound to {alpha}2M* at the same concentrations listed above. Cells were also treated with {alpha}2M* alone at a concentration of 500 nM or 500 pM for 1 h, washed, and then treated with CpG ODN at the concentrations listed above to assess the potential for {alpha}2M*-priming of immune cells.

{alpha}2M*-ODN complex stability
The stability of {alpha}2M*-ODN complexes was measured by generating and purifying complexes using the same methods described above. Complexes were separated from unbound CpG ODN so that free CpG ODN would not alter the equilibrium of spontaneous CpG ODN dissociation from {alpha}2M*. Purified complexes were incubated at 0°C and 37°C and analyzed by PAGE under native conditions. The amounts of bound and dissociated CpG ODN were detected by fluorescent imaging, as described previously. Samples incubated at 37°C were analyzed daily, and those incubated at 0°C were analyzed daily initially and then every 4–6 months for 18 months.

Rate of uptake
TG-elicited murine peritoneal M{Phi}s were obtained, plated, and washed as described above. Cells were treated with 0.5 µM-free or {alpha}2M*-bound CpG ODN and incubated at 37°C for durations of 1 min–2 h. Cells were washed four times with ice-cold isotonic solution: 150 mM NaCl, 25 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 0.5% BSA, pH 7.4 (Buffer A), to remove unbound ligands. Uptake of ligands was measured directly by fluorescent imaging as described previously.

To determine the length of exposure to CpG ODN required for cellular stimulation, TG-elicited murine peritoneal M{Phi}s were obtained, plated, and washed as described above. Cells were treated with free or {alpha}2M*-bound CpG ODN at a concentration of 0.5 µM and incubated for the durations ranging from 15 min to 4 h, after which time, cells were washed, resuspended in fresh media, and incubated for the remainder of 24 h from the initial exposure to CpG ODN. TNF was measured using a DuoSet ELISA kit from R&D Systems.

Mechanism of uptake
TG-elicited murine peritoneal M{Phi}s were obtained, plated, and washed by the same methods described above. Plates were moved to 4°C for 30 min to allow thermal equilibration. Cells were washed three times and cultured in ice-cold Buffer A. To measure nonspecfic binding, a portion of cells was washed and incubated in a similar buffer, except that it contained 5 mM EDTA and lacked Ca2+ and Mg2+ (Buffer B). {alpha}2M* was labeled using the infrared (IR) Dye 800CW protein labeling kit (Li-Cor Biosciences, Lincoln, NE, USA). Cells were treated with 1.0 nM IR-labeled {alpha}2M* and free CpG ODN or {alpha}2M*-ODN at increasing concentrations from 1.0 nM up to 100 nM. Cells were incubated at 4°C for 18 h and then washed four times with Buffer A; the cells incubated in Buffer B were washed with Buffer B. Surface-bound {alpha}2M* was measured directly by IR imaging using an Odyssey® IR imaging system (Li-Cor Biosciences). The amount of nonspecific binding accounted for less than 1% of the total binding in the absence of competing ligands.

To determine the effects of Ni2+ on the cellular binding of free CpG ODN and {alpha}2M*-ODN complexes, cells were treated with {alpha}2M*-ODN or free CpG ODN at a concentration of 0.5 µM, and NiSO4 was added over a range of concentrations, from 0.6 mM up to 10.0 mM. Cells were incubated at 4°C for 18 h and washed, and surface binding of free CpG ODN or {alpha}2M*-ODN was measured directly by fluorescent imaging as described previously.

Nuclease protection
TG-elicited murine peritoneal M{Phi}s were obtained, plated, and washed as described above. Prior to treating cells with free or {alpha}2M*-bound CpG ODN, these ligands were incubated with Benzonase or DNase I for 30 min at 37°C. These ligands were then added to cells at a concentration of 0.5 µM, and cells were incubated for 24 h at 37°C and 5% CO2. Media were collected, and TNF was measured by ELISA.

Enhanced stimulation of hPBMC
Approval of the following procedure was obtained from the Duke University Institutional Review Board; all experiments were performed in accordance with relevant guidelines and regulations. PBMC were obtained from healthy adult volunteers and isolated using a Ficoll-Hypaque density gradient, as described elsewhere [39 ]. Cells were washed three times and plated in 96-well plates at a concentration of 1 x 106 cells per well in RPMI 1640 with 5% human AB serum, L-glutamine, 20 mM HEPES, 1 mM sodium pyruvate, 100 µM MEM nonessential amino acids, 100 U/ml penicillin G, and 100 µg/ml streptomycin.

{alpha}2M*-ODN complexes were prepared by the same methods described above, except that ODN #2395 was used. Dose-response data were obtained by treating cells with equal amounts of free or {alpha}2M*-bound CpG ODN at the stated concentrations and incubating at 37°C and 5% CO2 for 24 h. The media were collected, and cytokines were measured using DuoSet ELISA kits (R&D Systems). Response kinetics were obtained by performing similar experiments, except that cells were treated with CpG ODN at a concentration of 0.5 µM. Media were collected at the time-points stated, and cytokines were measured by the same methods. As controls, cells were treated with {alpha}2M* alone and with a control sequence ODN bound to {alpha}2M* at the same concentrations listed above. Cells were also treated with {alpha}2M* alone at a concentration of 500 nM or 500 pM for 1 h, washed, and then treated with CpG ODN at the concentrations listed above to assess the potential for {alpha}2M* priming of immune cells.


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RESULTS
 
CpG ODN incorporation into {alpha}2M*
To determine whether CpG ODN incorporate into {alpha}2M and/or {alpha}2M*, fluorescently labeled CpG ODN were mixed with {alpha}2M, followed by addition of HNE to proteolytically activate {alpha}2M to its receptor-recognized form, {alpha}2M*. Samples were analyzed by PAGE under native conditions; CpG ODN were detected by fluorescent imaging (Fig. 1C) , and {alpha}2M was detected by staining the gel with Coomassie brilliant blue (Fig. 1D) . As controls, samples of CpG ODN alone and a mixture of CpG ODN with {alpha}2M that did not contain HNE were analyzed separately. Figure 1C demonstrates that CpG ODN binds to receptor-recognized {alpha}2M* but not to {alpha}2M in its native conformation. In Lane 2, 96% of the CpG ODN present migrated as free CpG ODN, and only 4% bound to the protein, as evidenced by the faint band at the top of the lane corresponding to the location of {alpha}2M (Fig. 1D) . In Lane 3, however, 87% of the CpG ODN present was bound to {alpha}2M*, which had been converted to its receptor-recognized form by HNE. Based on these results, the molar ratio of CpG ODN bound to {alpha}2M* is calculated as ~4.5 moles CpG ODN/mole {alpha}2M*. Although {alpha}2M and {alpha}2M* can be distinguished from each other by PAGE analysis under native conditions, such analysis cannot be done on this gel, as the conditions used were chosen to optimize the separation of free CpG ODN from both forms of {alpha}2M while allowing free CpG ODN to remain on the gel. Separation of {alpha}2M from {alpha}2M* requires a longer period of electrophoresis, which results in the loss of unbound CpG ODN from the gel. These samples were also analyzed under conditions that separate {alpha}2M from {alpha}2M* to confirm that the {alpha}2M had undergone complete activation by HNE (data not shown).

After establishing that CpG ODN preferentially bind to {alpha}2M* but not native {alpha}2M, specificity of the binding was examined. Incorporation reactions with {alpha}2M and labeled CpG ODN were carried out in the presence of increasing amounts of unlabeled CpG ODN. Samples were analyzed by PAGE under native conditions, and bound CpG ODN were measured by fluorescent imaging (Fig. 2A ). In the presence of fivefold molar excess unlabeled CpG ODN, the amount of labeled CpG ODN bound is reduced by 60%, and this amount is reduced by 95% in the presence of a 100-fold molar excess of unlabeled CpG ODN (Fig. 2C) . These data demonstrate competition between the two ligands, suggesting that CpG ODN bind to {alpha}2M* in a specific, saturable manner that is independent of the fluorescent label. These experiments were performed using P = O and P = S CpG ODN; similar results were obtained.


Figure 2
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  		Figure 2. CpG ODN bind to {alpha}2M* in a specific, saturable manner. {alpha}2M was mixed with a fixed amount of labeled CpG ODN and increasing amounts of unlabeled CpG ODN. Numbers 0–100 indicate the molar ratio of unlabeled to labeled CpG ODN. (A) {alpha}2M*-bound CpG ODN were detected by fluorescent imaging. (B) {alpha}2M*-ODN complexes were detected by Coomassie brilliant blue staining. (C) Signals from A were measured, and amounts are displayed graphically. Data are means of the experiment performed in triplicate; error bars indicate ± SD.

Enhanced biological activity of CpG and {alpha}2M*-ODN
We next sought to determine how incorporation into {alpha}2M* affects the immunostimulatory properties of CpG ODN. M{Phi}s, which play a key role in the murine response to CpG ODN by secreting proinflammatory cytokines that recruit and activate other immune cells [20 , 40 ], were selected as the model in which to first measure the biological activity of {alpha}2M*-ODN complexes. Murine M{Phi}s were treated with free or {alpha}2M*-bound CpG ODN over a range of concentrations, and biological activity was measured initially by quantifying the production of TNF, a classic TH1 cytokine. Over the entire dose-range tested, CpG ODN bound to {alpha}2M* exhibit an enhanced ability to stimulate M{Phi}s compared with free CpG ODN (Fig. 3A ). {alpha}2M* increases the potency of CpG ODN by four- to sixfold, and the most pronounced enhancement occurs at the lower concentrations of CpG ODN tested. From concentrations of 0.13–1.0 µM, the average increase in efficacy is greater than fivefold for {alpha}2M*-bound versus free CpG ODN.


Figure 3
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  		Figure 3. Incorporation in {alpha}2M* enhances the ability of CpG ODN to activate murine cells. Murine M{Phi}s were treated with equal amounts of free or {alpha}2M*-bound CpG ODN, and TNF production was measured by ELISA. (A) Free or bound CpG ODN were administered over a range of concentrations, and TNF was measured after 24 h. A portion of cells were pre-treated with {alpha}2M* prior to administration of free CpG ODN. {alpha}2M*-ODN ({blacksquare}); free CpG ODN ({square}); 500 pM/{alpha}2M* pre-treatment ({blacksquare}), and 500 nM {alpha}2M* pre-treatment ({blacksquare}) followed by free CpG ODN at the state concentration. Next, cells were treated with CpG ODN at a concentration 0.5 µM, and cytokines were measured over several time points. Those measured included (B) TNF; (C) IL-1{alpha}; (D) IL-6; (E) IL-12 (p70); and (F) MCP-1. Data are a representative example from one of three experiments, each performed in triplicate; error bars indicate ± SD. *, P < 0.05; **, P < 0.005, between {alpha}2M*-ODN and all other groups, as calculated by the Student’s t-test.

In control studies, cells were treated with {alpha}2M* complexes that contained a GpC ODN control sequence and with {alpha}2M* alone at the same concentrations as those used in the previous experiment; both failed to elicit any response that could be detected at all concentrations tested (data not shown). These negative results demonstrate that {alpha}2M*-ODN complexes stimulate cells through the same CpG-dependent mechanism through which free CpG ODN stimulate cells. They demonstrate further that {alpha}2M* alone is not responsible for the immune system activation. The {alpha}2M *-ODN complexes used were all free of LPS contamination, as determined by the QCL assay; negative results obtained in this biological assay confirm that {alpha}2M* alone and {alpha}2M*-ODN complexes are free of possible contaminants that might elicit an immune response. Cells were also treated with equal concentrations of labeled and unlabeled CpG ODN. Over a dose range similar to that tested previously, there was no difference in the amount of TNF produced in response to stimulation by labeled CpG ODN compared with the response elicited by unlabeled CpG ODN (data not shown), confirming that the fluorescent label affects neither the potency nor efficacy of CpG ODN.

The experiment was repeated using cells harvested from TLR9-KO mice. Cells were treated with equal amounts of free or {alpha}2M*-bound CpG ODN, and the amount of TNF produced was measured by ELISA; the results are shown in Table 1 . Similar to previous experiments, CpG ODN bound to {alpha}2M* elicited responses that were much greater than those produced by equal amounts of free CpG ODN. In addition, the ability of cells to respond to free or {alpha}2M*-bound CpG ODN was dependent on the presence of TLR9. Cells from the KO mice, however, still exhibited the ability to produce an innate immune response, as evidenced by their response to LPS. These results demonstrate that the mechanism by which {alpha}2M*-bound CpG ODN stimulate cells is the same as the mechanism by which free CpG ODN stimulate cells.


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  		Table 1. TNF Production by {alpha}2M*-ODN Complexes Is Dependent on TLR9

The stability of {alpha}2M*-ODN complexes was measured to verify that they remained intact long enough to be taken up by cells and exert the enhanced biological activity observed. P = O and P = S {alpha}2M*-ODN complexes were purified and incubated at 37°C and 0°C and analyzed by PAGE; {alpha}2M*-bound and dissociated CpG ODN were measured by fluorescent imaging. At 37°C, 80% of P = O and P = S {alpha}2M*-ODN complexes remain intact after 24 h, and they exhibit a half-life of 96 h. When samples were incubated at 0°C, 95% of both types of complex remained intact after a period of 18 months. Based on these findings, the vast majority of complexes remains intact long enough to be taken up by cells and exert their effects within the time span of these experiments.

Another potential mechanism for the enhanced response to {alpha}2M*-ODN complexes is {alpha}2M*-induced signaling. In addition to the {alpha}2M* endocytic receptor LRP/CD91, M{Phi}s express on their surface the {alpha}2M*-signaling receptor, glucose-regulated protein-78 (GRP78/BiP). On binding GRP78, {alpha}2M* initiates changes in calcium levels, cAMP, and inositol triphosphate [41 ]. It is possible that such signaling, induced by {alpha}2M*, might prime cells to respond, making them more responsive to CpG ODN that are present. If GRP78 signaling were involved in the response, pretreatment with {alpha}2M* alone followed by treatment with CpG ODN should demonstrate similar enhancement to that observed previously with {alpha}2M*-ODN complexes. To determine whether GRP78 signaling affects the activity of {alpha}2M*-ODN complexes, M{Phi}s were pretreated with {alpha}2M* for 1 h and washed; CpG ODN were then added with fresh media, and TNF production was measured by ELISA. The {alpha}2M* concentrations used were 500 pM, which is tenfold greater than the Kd of GRP78 for {alpha}2M* and induces maximal signaling [42 ], and 500 nM, the {alpha}2M* concentration occurring in the highest dose of {alpha}2M*-ODN complexes tested. Neither concentration of {alpha}2M* had an effect on the CpG ODN-induced response (Fig. 3A) , indicating that the {alpha}2M*-GRP78 signaling pathway does not contribute to the augmented response elicited by {alpha}2M*-ODN complexes.

After finding that {alpha}2M*-bound CpG ODN exhibit enhanced potency and efficacy for immunostimulation, the kinetics of the responses induced by free and bound CpG ODN was compared by measuring immune stimulation over time. M{Phi}s were treated with equal amounts of free or {alpha}2M*-bound CpG ODN, and TNF production was measured over five time-points (Fig. 3B) . The most pronounced differences between the responses occur early; the response to {alpha}2M*-ODN complex is detected after 2 h, the first time-point tested, and that induced by free CpG ODN is not detectable until after 8 h, by which time the response to {alpha}2M*-ODN has reached its peak. The peak response to {alpha}2M*-ODN is not only twice that induced by free CpG ODN, but it also occurs 16 h earlier.

CpG ODN stimulate M{Phi}s to produce several cytokines and chemokines in addition to TNF, including IL-1, IL-6, IL-12, and MCP-1 [43 , 44 ]. These effector molecules are crucial to the development of the TH1-biased response that is characteristic of CpG ODN stimulation. To characterize more fully the immune response elicited by {alpha}2M*-ODN complexes, the production of these cytokines over time was measured using multiplex analysis (Fig. 3C 3D 3E) . Again, the differences in cytokine production between free and bound CpG ODN stimulation are most pronounced at the earlier time-points. Complex-induced responses begin and peak earlier and with the exception of IL-6, reach levels that are at least twofold greater than those induced by free CpG ODN. {alpha}2M*-bound CpG ODN demonstrate an enhanced ability to stimulate the production of numerous cytokines that are central to a TH1-biased immune response. Similar to results obtained with respect to TNF responses, there were no responses to the negative controls {alpha}2M* alone or the control sequence ODN bound to {alpha}2M*, and there was also no enhancement by pretreatment with {alpha}2M* followed by CpG ODN (data not shown).

Rate of uptake
Previous studies demonstrated that {alpha}2M*, along with incorporated molecules, binds to its uptake receptor, LRP, and is taken up rapidly; the circulating half-life of {alpha}2M* is 2–5 min in plasma, and the primary receptor involved in plasma clearance is LRP on Kuppfer cells and hepatocytes of the liver [31 , 45 ]. Based on the efficiency with which {alpha}2M* complexes are taken up by receptor-bearing cells, we hypothesized that such rapid uptake is at least partially responsible for the enhanced biological activity of {alpha}2M*-bound CpG ODN. To compare the rates of uptake of {alpha}2M*-ODN complexes and free CpG ODN, murine M{Phi}s were treated with equal amounts of free or {alpha}2M*-bound CpG ODN and incubated at 37°C. After periods from 1 min up to 2 h, cells were washed to remove unbound ligands. Bound and internalized CpG ODN were measured directly by fluorescent imaging as shown in Figure 4A . Uptake of all four ligands, free and {alpha}2M*-bound P = O and P = S CpG ODN, follows a linear pattern of uptake over time, but those bound to {alpha}2M* are taken up significantly faster. P = O and P = S ODN bound to {alpha}2M* are taken up at a sixfold greater rate than free P = S CpG ODN and at a 50-fold greater rate than free P = O CpG ODN. These results show that the rate of {alpha}2M*-ODN uptake is not affected by the backbone composition of the incorporated CpG ODN and that CpG ODN bound to {alpha}2M* are internalized by immune-competent cells significantly faster than free CpG ODN.


Figure 4
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  		Figure 4. {alpha}2M* increases the biological activity of CpG ODN by increasing their rate of cellular uptake. (A) Murine M{Phi}s were treated with free or {alpha}2M*-bound CpG ODN for durations of 1 min up to 2 h, after which time, cell were washed to remove unbound ligands. The amount of CpG ODN taken up was measured directly by fluorescent imaging. (B) Murine M{Phi}s were treated with free or bound CpG ODN for durations of 15 min up to 4 h and then washed. Fresh media were added, and the cells were incubated for the remainder of 24 from the time of initial CpG ODN exposure; TNF production was measured by ELISA. {alpha}2M*-ODN ({blacksquare}; P=S); {alpha}2M*-ODN ({blacktriangleup}; P=O); free CpG ODN ({square}amp;; P=S); free CpG ODN ({triangleup}; P=O). Data are a representative example from one of three experiments, each performed in triplicate; error bars indicate ± SD. *, P < 0.05; and **, P < 0.005, as calculated by the Student’s t-test. (A) **, Both {alpha}2M*-ODN data plots compared with both free CpG ODN data plots.

Based on the high uptake rate of the {alpha}2M*-ODN complex compared with that of free CpG ODN, we hypothesized that CpG ODN-responsive cells would be stimulated more rapidly by CpG ODN bound to {alpha}2M* than by free CpG ODN. Murine M{Phi}s were treated with 0.5 µM free CpG ODN or CpG ODN bound to {alpha}2M*. After periods of exposure ranging from 15 min to 4 h, the cells were washed to remove unbound ligands. Fresh media were added, and TNF production was measured 24 h after the initial exposure to CpG ODN. As Figure 4B shows, cells are stimulated to produce a measurable response with as little as 15 min of exposure to {alpha}2M*-ODN, the shortest duration tested, and it takes 2 h of exposure to free CpG ODN to elicit a response, by which time cells exposed to {alpha}2M*-ODN have produced a response that is eightfold greater than that induced by free CpG ODN. Based on these results, rapid uptake is one mechanism by which incorporation into {alpha}2M* enhances the biological response to CpG ODN. As shown in Figure 3A , there is graded production of cytokines in response to increasing amounts of CpG ODN. It follows then that the more CpG ODN that are internalized by cells, the greater the immune response will be. As CpG ODN bound to {alpha}2M* are endocytosed at a greater rate than free CpG ODN, it is likely that the minimum amount of internalized CpG ODN necessary to elicit an immune response will be reached more quickly when CpG ODN are bound to {alpha}2M*.

Mechanism of uptake
Previous studies investigating the behavior of {alpha}2M*-protein complexes have shown that these complexes are taken up exclusively through LRP-mediated endocytosis and that the interaction between {alpha}2M* and LRP is unaltered by the incorporation of proteins [30 , 31 ]. We hypothesize that the {alpha}2M*-LRP interaction is likewise unaffected by the incorporation of CpG ODN, but this is an extrapolation of prior data from studies about {alpha}2M* protein complexes. To determine whether the interaction between {alpha}2M* and LRP is affected by the incorporation of CpG ODN, binding studies were performed. Murine M{Phi}s were treated with 1.0 nM IR-labeled {alpha}2M* and increasing concentrations of free or {alpha}2M*-bound CpG ODN. Cells were incubated at 4°C for 18 h and then washed to remove unbound ligands. Bound {alpha}2M* was measured directly by IR imaging; the results are shown in Figure 5A . As predicted, P = O and P = S {alpha}2M*-ODN complexes demonstrate the ability to compete with {alpha}2M* for surface binding, and free CpG ODN shows no effect on {alpha}2M* binding. Based on the similar binding affinities of {alpha}2M* and {alpha}2M*-ODN, it is evident that CpG ODN incorporation, regardless of the CpG ODN backbone composition, does not alter the receptor-mediated endocytosis of {alpha}2M* by LRP.


Figure 5
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  		Figure 5. Incorporation into {alpha}2M* confers protection from nuclease digestion but does not hinder the binding of {alpha}2M* to its uptake receptor. (A) Murine M{Phi}s were treated with 1.0 nM-labeled {alpha}2M* and increasing concentrations of free CpG ODN or {alpha}2M*-ODN. Cells were incubated at 4°C for 18 h and then washed to remove unbound {alpha}2M*; surface-bound {alpha}2M* was measured directly by IR imaging. (B) Murine M{Phi}s were treated with free or {alpha}2M*-bound CpG ODN and increasing concentrations of NiSO4. Cells were incubated at 4°C for 18 h and then washed to remove unbound ligands. Surface-bound ligands were measured directly by fluorescent imaging. (C) Free or {alpha}2M*-bound CpG ODN were incubated with nucleases Benzonase or DNase I prior to their addition to cells. After 24 h, TNF production was measured by ELISA. {alpha}2M*-ODN ({blacksquare}; P=S); {alpha}2M*-ODN ({blacktriangleup}; P=O); free CpG ODN {square}amp;; P=S); free CpG ODN ({triangleup}; P=O). Data are a representative example from one of three experiments, each performed in triplicate; error bars indicate ± SD. (C) **, P < 0.005, each in comparison with response measured from free CpG ODN without nuclease treatment, as calculated by the Student’s t-test.

A second method was used to confirm that {alpha}2M*-ODN are taken up by LRP. The surface binding of these ligands was measured in the presence of increasing concentrations of Ni2+, a metal ion that inhibits the interaction between {alpha}2M* and its receptor LRP [46 ]; it should be noted that no studies to date have reported the effects of Ni2+ on cellular binding of free CpG ODN. Murine M{Phi}s were treated with 0.5 µM-free or {alpha}2M*-bound CpG ODN and increasing concentrations of Ni2+. Cells were incubated at 4°C for 18 h and washed to remove unbound ligands. Bound CpG ODN were measured by fluorescent imaging. Nonspecific binding was assessed by incubating cells in the same solutions, except that they contained the chelating agent 5 mM EDTA instead of 1 mM Ca2+ or Mg2+, which is necessary for the binding of {alpha}2M* [45 ] and free CpG ODN [47 , 48 ].

As reported previously, {alpha}2M* binding to cells is inhibited by the presence of Ni2+, but CpG ODN surface binding is actually enhanced by Ni2+ (Fig. 5B) . Similar to the binding studies discussed earlier, these results demonstrate that the incorporation of CpG ODN into {alpha}2M* does not alter the cellular binding of {alpha}2M*. These {alpha}2M*-ODN complexes bind LRP with the same affinity as {alpha}2M* alone, and this binding is inhibited by Ni2+ in the same manner. Although the purpose of this experiment was to characterize the surface binding of {alpha}2M*-ODN complexes further, we also discovered that free CpG ODN binding is positively affected by the presence of Ni2+, a phenomenon that had not been described previously.

Nuclease protection
Proteins incorporated within {alpha}2M* are blocked from interacting with other molecules in solution. Interactions are blocked between antibodies and specific ligands, signaling molecules and their receptors, and enzymes and substrates [30 , 31 ], a quality that first characterized {alpha}2M as a proteinase inhibitor. Based on the characteristics of {alpha}2M* complexes, we hypothesized that {alpha}2M* may confer protection from nuclease digestion to incorporated CpG ODN. Free and {alpha}2M*-bound P = O CpG ODN were incubated with the nucleases Benzonase and DNase I and then added to murine M{Phi}s. Media were collected after incubation for 24 h, and responses were measured. The raw data were normalized so that responses to the CpG ODN not preincubated with nucleases were assigned values of 100, and the remaining responses were adjusted accordingly (Fig. 5C) . Although both nucleases reduce the activity of free CpG ODN by 75%, they have no effect on the activity of CpG ODN bound within {alpha}2M*, indicating that {alpha}2M* protects CpG ODN from inactivation by nuclease digestion.

These data are the first to establish the CpG ODN-binding site on {alpha}2M*. This nuclease protection is likely the result of steric hindrance between the bound CpG ODN and nucleases present in solution. As {alpha}2M* undergoes proteolytic conversion to its receptor-recognized form, there is a major conformational change that reduces the size of the binding pocket openings, thus preventing molecules larger than 20 kDa from diffusing into or out of the binding pocket [30 , 31 ]. As CpG ODN are not digested by nucleases, they are likely trapped within the {alpha}2M*-binding pocket, so named for its tendency to bind numerous protein ligands [30 ]. Small nucleases that are capable of diffusing into the binding pocket may still be inhibited, as CpG ODN may incorporate into {alpha}2M* in a manner that shields the backbone, making it inaccessible to nucleases. Likewise, the dimensions of the pocket may limit the movement and orientation of nucleases, precluding proper alignment of the enzyme active site with the CpG ODN backbone.

As a result of the synthetic nature of the in vitro cell culture system, there are likely few nucleases present that could inactivate P = O CpG ODN, which is why P = O CpG ODN are only slightly less potent than their P = S counterparts compared with drastic potency differences in vivo. Based on the extremely short half-life and efficacy of P = O CpG ODN compared with P = S equivalents in vivo, however, natural biological situations likely have a much higher level of nuclease activity. This suggests that {alpha}2M*-bound P = O CpG ODN will demonstrate even greater enhancement in vivo than that observed through in vitro testing.

Enhanced stimulation of hPBMC
The enhanced biological activity of {alpha}2M*-ODN complexes demonstrated by activation of murine M{Phi}s might not occur with human cells, which show numerous differences in the biological activity of CpG ODN. The TLR9 proteins expressed by human and murine cells share a large portion of sequence identity, but each recognizes different CpG motifs that optimally stimulate responses [25 , 49 , 50 ]. In mice, the motif shown to be most active is 5'-Pu-Pu-CpG-Py-Py-3', such as those found in the ODN #1826. In humans, there are three classes of CpG ODN, which stimulate distinctly different subsets of cells to generate qualitatively different responses. Class A (D type) ODN, composed of a palindromic P = O middle segment capped with P = S-modified ends, activate NK cells and pDCs to produce IFN-{gamma} and IFN-{alpha}, respectively [16 , 51 , 52 ], but fail to stimulate B cells. Class B (K type) ODN are wholly P = S sequences with multiple CpG motifs and a poly(G) tail at the 3' end; this class triggers IgM and IL-6 production from B cells, activates pDCs to produce TNF rather than IFN-{alpha}, and stimulates strong, cytolytic activity in NK cells [37 , 38 , 51 ]. Class C ODN is a hybrid of the first two classes with a completely P = S-modified backbone, palindromic sequences, but no poly(G) tail; this class has the ability to induce type I IFN production as well as B cell activation [38 ]. Another key difference is the cellular expression of TLR9; human M{Phi}s do not express TLR9 and are therefore unresponsive to CpG ODN. Rather, the human cells activated by CpG ODN include pDCs, B cells, and NK cells [53 ].

To demonstrate the ability of {alpha}2M*-ODN complexes to stimulate an immune response in humans, PBMC were treated with CpG ODN #2395, a Class C ODN that exhibits optimal activity in stimulating PBMC [38 ]. Cells were treated with equal amounts of free or {alpha}2M*-bound CpG ODN over a range of concentrations and incubated for 24 h; media were collected, and cytokines were measured by ELISA. The production of IFN-{alpha}, IL-6, and TNF is shown in Figure 6A 6C and 6E , respectively, which demonstrates that {alpha}2M* incorporation enhances the potency and efficacy of CpG ODN in hPBMC. The amount of IFN-{alpha} produced in response to 0.25 µM {alpha}2M*-ODN is tenfold greater than that induced by the same amount of free CpG ODN. The maximum amount of IFN-{alpha} in response to {alpha}2M*-ODN is nearly twice that of the maximal response to free CpG ODN. Although the peak IL-6 responses are similar, the maximal response to {alpha}2M*-ODN is elicited at a fourfold lower dose than free CpG ODN. TNF production elicited by complexes is greater at every concentration tested and is twofold greater at all but the highest CpG ODN concentration. As controls, PBMC were treated with {alpha}2M* alone and {alpha}2M* carrying an ODN control sequence with no biological activity. At concentrations similar to those used in the previous experiment, responses to {alpha}2M* alone and control sequence {alpha}2M*-ODN complexes were undetectable (data not shown). These findings confirm that the {alpha}2M* itself is not responsible for the immune response and that cellular activation is dependent on the CpG motif of the ODN incorporated within {alpha}2M*. The data confirm further that the complexes used to elicit immune responses are free of contamination by other agents that might cause an immune response, such as LPS.


Figure 6
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  		Figure 6. {alpha}2M*-ODN complexes exhibit an enhanced ability to stimulate human cells. (A) hPBMC were treated with free or {alpha}2M*-bound CpG ODN over a range of concentrations, and cytokine production was measured in the media after 24 h; those measured include (A) IFN-{alpha}, (C) IL-6, and (E) TNF. Cells were treated with 0.5 µM-free or {alpha}2M*-bound CpG ODN, and cytokine production was measured after several durations; (B) IFN-{alpha}, (D) IL-6, and (F) TNF. Data are a representative example from one of three experiments, each performed in triplicate; error bars indicate ± SD. *, P < 0.05, and **, P < 0.005, for each pair of data, as calculated by the Student’s t-test.

The response kinetics of PBMC was also examined. PBMC were treated with 0.5 µM-free or {alpha}2M*-bound CpG ODN and incubated for varying durations; cytokines were measured by ELISA. Figure 6B 6D and 6F , shows the production of IFN-{alpha}, IL-6, and TNF, respectively, over a period of 48 h. Like the previous dose-response curves, these figures show the superior efficacy of {alpha}2M*-ODN over free CpG ODN; the peak production of IFN-{alpha} is enhanced by twofold, that of IL-6 is enhanced by sixfold, and the maximum TNF production is increased by 15-fold. In addition to greater peak responses, the responses elicited by {alpha}2M*-ODN begin much earlier than those induced by free CpG ODN stimulation. IFN-{alpha} production reached a level of 700 pg/ml just 8 h after stimulation with {alpha}2M*-ODN; this level was not reached by free CpG ODN activation until 24 h after treatment. Similarly, {alpha}2M*-ODN stimulated the production of 750 pg/ml IL-6 within 4 h, and free CpG ODN treatment did not reach that level of IL-6 production until after 48 h. The differences in TNF production are the most striking; the responses to free and {alpha}2M*-bound CpG ODN peak after 8 h, but with 15-fold greater production of TNF from those cells treated with {alpha}2M*-ODN, the response to free CpG ODN is barely visible on the same scale.

These data expand our knowledge of the immunostimulatory properties of {alpha}2M*-ODN complexes. In addition to their similarity to our findings with murine M{Phi}s, these results confirm that {alpha}2M* greatly enhances the ability of CpG ODN to stimulate immune responses in human cells. Despite numerous differences in TLR9 biology between mice and humans, {alpha}2M*-bound CpG ODN consistently demonstrate enhanced potency and efficacy for activation of immune-competent cells, with the responses beginning and peaking earlier.


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DISCUSSION
 
The interaction between CpG ODN and numerous plasma proteins has been described [54 55 56 57 58 ]. Although previously, documented interactions involve the abrogation of biological activity of CpG ODN, here, we report the first evidence of an interaction between CpG ODN and a plasma protein that not only maintains CpG ODN immunostimulatory properties but also, actually enhances them. The binding of CpG ODN to {alpha}2M* is specific and saturable, and the ratio of incorporation is ~4.5 moles CpG ODN/mole {alpha}2M*. We have shown CpG ODN bound to {alpha}2M* stimulate responses from immune-competent cells, and these responses are dependent on the presence of TLR9. Using murine and human cells, we have demonstrated that binding to {alpha}2M* enhances the potency and efficacy of CpG ODN. By increasing the rate of CpG ODN uptake by sixfold, {alpha}2M*-aided delivery leads to an immune response that begins and peaks earlier than that induced by free CpG ODN.

These results are promising in light of the various proposed clinical applications of CpG ODN; we predict that the in vivo immunostimulatory activity of CpG ODN bound to {alpha}2M* will be enhanced even more than what we have observed in vitro. The in vitro models used are optimized to detect a response to CpG ODN; cells in tissue culture are in a closed, static system with CpG ODN in constant contact with the cells and a relative absence of nucleases. The in vivo model, however, offers many more obstacles to CpG ODN-induced immunostimulation. CpG ODN can be degraded by nucleases, bind serum proteins nonspecifically, or be taken up by nonresponsive cells. However, CpG ODN incorporated within {alpha}2M* adopt the pharmacokinetics of {alpha}2M* and so are targeted for receptor-mediated uptake by M{Phi}s, DCs, and B cells, the cell types that are crucial to the CpG ODN-induced immune response. Based on the protection and targeted delivery that {alpha}2M* packaging offers, many of the in vivo obstacles to CpG ODN efficacy may be overcome.

{alpha}2M*-aided delivery may offer advantages over the alternate methods used for improving the activity of CpG ODN. Packaging CpG ODN into liposomes yields greater immune responses; however, liposomes are taken up predominantly by cells nonspecifically. In addition, CpG-independent immune stimulation and a high level of toxicity often associated with liposomes pose significant barriers to their use as a delivery vehicle for CpG ODN [59 , 60 ]. Packaging CpG ODN into virus-like particles (VLP) has also been used to improve CpG ODN efficacy. Although this method targets CpG ODN for uptake by immune-competent cells and protects them from nuclease digestion [61 ], the use of VLP has its own drawbacks. The use of viral proteins could interfere with the specificity of the desired immune response, and repeated dosing could break tolerance. In addition, high production costs of VLP could limit their widespread use.

Nuclease resistance has particular relevance in the context of therapeutics. P = O CpG ODN are degraded rapidly in vivo, impairing their ability to stimulate an adequate response [62 , 63 ]. P = S modification of the backbone confers nuclease resistance but alters the immune response [21 , 24 , 25 ] and increases nonspecific binding to plasma proteins and cell surfaces, which can have disruptive effects [28 , 29 ]. Incorporation of CpG ODN into {alpha}2M* prevents nuclease digestion and nonspecific interactions, thus permitting the clinical use of P = O or P = S CpG ODN. This may be advantageous, as CpG ODN with different backbone structures elicit intrinsically different immune responses by activating different cell types [16 , 38 , 64 ]. Free of backbone limitations, the repertoire from which to design CpG ODN is expanded greatly, hopefully yielding greater success at tailoring CpG ODN to induce responses that can best combat specific diseases.

Although the plasma half-life of {alpha}2M* is short (2–5 min) [31 , 45 ], if {alpha}2M*-ODN complexes were developed as therapeutics, they would not be administered i.v. but most likely by intramuscular, s.c., or intradermal injection or transdermally to take advantage of the highly efficient APC that are present in the skin and draining lymph nodes. In peripheral tissue, {alpha}2M* complexes are taken up predominantly by APC, Langherhans cells, DCs, and M{Phi}s, which are responsible for the adaptive immune response that ensues following {alpha}2M*-aided delivery of antigens [65 ]. As DCs also play a major role in the immune response to CpG ODN, we predict that with nonvascular administration of {alpha}2M*-ODN complexes, CpG ODN will be targeted for receptor-mediated uptake by peripheral DCs, and we will observe vastly superior potency and efficacy of CpG ODN over what has been observed previously.

Other strategies have been used to improve the immunostimulatory properties of CpG ODN. Studies have shown that CpG ODN which are chemically conjugated to an antigen elicit a greater immune response than that elicited by free CpG ODN mixed with antigen [66 ]. The explanation for this synergistic effect is that the antigen and CpG ODN colocalize to the same cells and that conjugation facilitates the uptake of antigens. Similar enhanced responses may be observed following simultaneous treatment with {alpha}2M*-ODN and {alpha}2M* antigen complexes; APC have sufficient amounts of receptors for {alpha}2M*, and simultaneous treatment with these two types of {alpha}2M* complexes will almost certainly assure that the CpG ODN and antigen are delivered to the same endocytic compartments in the same target cells. Furthermore, we are currently in the process of generating {alpha}2M* complexes that contain CpG ODN and a protein antigen. Such complexes will ensure the simultaneous delivery of CpG ODN and antigen to the same cells and intracellular compartments via high-affinity, receptor-dependent uptake while protecting each from nucleases and proteases, respectively. The data collected about the enhanced responses to CpG ODN-conjugated antigens raise the concern that the coupling of CpG ODN to {alpha}2M* could induce a response to {alpha}2M* itself. There have been no documented cases of immune reactions to this endogenous plasma protein, and no reported immunologically relevant isoforms have been identified. However, the association of CpG DNA with targets of autoimmune reactions has been documented [67 ]. These autoantigens tend to be isolated from the immune system by their intracellular location, whereas {alpha}2M is constitutively present in plasma at micromolar concentrations [31 ], so irregular contact between {alpha}2M and immune-competent cells as a result of therapeutic administration is not a likely trigger for autoimmunity. Still, the link between CpG DNA and autoimmune reactions warrants examination of this possibility with the use of {alpha}2M* as a delivery vehicle for CpG ODN-based therapeutics.

{alpha}2M*-aided delivery of CpG ODN could be used as a tool to further examine the mechanisms of CpG ODN-induced immune stimulation. The characteristics of the immune responses elicited by CpG ODN are dependent on numerous variables, including the motifs surrounding the CpG dinucleotide, sequences, and backbone modifications [16 , 38 , 51 ]. Immune stimulation is initiated by CpG binding to TLR9, which must be preceded by cellular uptake and trafficking to the endosome. These steps are influenced by CpG ODN sequence, length, and backbone modifications [24 , 64 , 68 69 70 ]. As a result of the overlaps, it has been difficult to discern whether the influences of sequence and backbone composition on immune responses are the result of altered uptake and trafficking or if they stem from a distinctly different interaction with TLR9 or some other downstream event. By packaging CpG ODN of various sequences and backbone compositions into {alpha}2M*, the differences in cellular uptake and interactions with extracellular proteins and nucleases will be eliminated, allowing for the unclouded examination of how these factors affect the nature of the immune responses. In addition, by treating purified B cells and pDC with {alpha}2M*-ODN complexes, we may further our understanding of the cell type-specific responses induced by CpG ODN in the absence of cell type-specific differences in the uptake and trafficking of CpG ODN. Greater comprehension of the mechanisms of response regulation will likely facilitate the design and delivery of immune system modulators with improved clinical efficacy.

{alpha}2M* packaging may be a valuable approach for the eventual clinical applications of CpG ODN, which have received significant attention as potential therapeutics and adjuvants against infectious diseases, cancer, and immune disorders. They have shown great promise thus far, and clinical trials are currently underway. Given the enhanced biological activity that {alpha}2M*-ODN complexes exhibit, clinical evaluation of such complexes may prove fruitful. Greater potency and targeted delivery to immune-competent cells may translate into smaller and fewer doses, which will reduce side-effects and cost. Superior efficacy as an adjuvant may lead to a more robust, primary-immune response that yields a higher rate of protective immunity or cancer immunosurveillance. The ability to develop a response faster and with fewer doses may be critical for developing vaccines or treatments for high-risk individuals or to reduce the impact of biological weapons. As the complexes protect CpG ODN from breakdown and facilitate uptake, they may likewise yield a superior pharmacokinetics profile, again resulting in greater efficacy with reduced side-effects.


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ACKNOWLEDGEMENTS
 
This study was funded by the National Heart, Lung, and Blood Institute, HC-24066. The authors thank Sturgis Payne, Dr. Steven Kaczowka, Steve Conlon, and Marie Thomas for their contributions to this work.

Received April 20, 2007; revised August 6, 2007; accepted August 30, 2007.


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