Specificity and immunochemical properties of anti-DNA antibodies induced in normal mice by immunization with mammalian DNA with a CpG oligonucleotide as adjuvant

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Clinical Immunology 109 (2003) 278 –287

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Specificity and immunochemical properties of anti-DNA antibodies induced in normal mice by immunization with mammalian DNA with a CpG oligonucleotide as adjuvant Trinh T. Tran,a Charles F. Reich III,b Munir Alam,c and David S. Pisetskya,b,d,* a

Division of Rheumatology, Duke University Medical Center, Durham, NC 27709, USA b Durham Veterans Administration Hospital, Durham, NC 27705, USA c Human Vaccine Institute, Duke University Medical Center, NC 27709, USA d Durham VA Medical Center, P.O. Box 151G, 508 Fulton Street, Durham, NC 27705, USA Received 5 June 2003; accepted with revision 1 August 2003

Abstract To elucidate the role of DNA antigen drive in the anti-DNA response, the specificity and immunochemical properties of anti-DNA antibodies induced in normal mice by immunization with double stranded (ds) mammalian DNA with a CpG oligonucleotide (ODN) adjuvant were characterized. Like spontaneous anti-DNA from MRL/lpr mice, the induced anti-DNA bound cross-reactively to DNA from five different species by ELISA. The induced antibodies displayed a predominance of IgG2a and had much lower amount of IgG3 than spontaneous antibodies. Surface plasmon resonance indicated that the induced and spontaneous anti-DNA antibodies have a similar range of avidity and binding kinetics. While sera from the MRL/lpr mice had substantial binding to histones and nucleosomes, the immunized mice had antibody levels to these antigens similar to those of mice treated only with incomplete Freund’s adjuvant. Together, these results indicate that normal mice can produce autoantibodies to dsDNA, with a CpG ODN allowing the generation of antibodies resembling those in spontaneous autoimmunity. © 2003 Elsevier Inc. All rights reserved. Keywords: Systemic lupus erythematosus; Anti-DNA; CpG oligonucleotide; Nucleosome; Adjuvant; Mammalian DNA

Introduction Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and are markers of diagnostic and prognostic significance. These antibodies target sites present on single stranded (ss) and double stranded (ds) DNA, with those directed to dsDNA closely linked to disease pathogenesis [1,2]. As demonstrated most compellingly by the study of monoclonal antibodies, antiDNA antibodies bear features of selection by DNA antigen [3–5]. While DNA in vivo likely exists in the form of nucleosomes released from dead or dying cells, the properties of this antigen have remained obscure and it has been difficult to model this response by immunization with either * Corresponding author. Fax: ⫹1-919-286-6891. E-mail address: [email protected] (D.S. Pisetsky). 1521-6616/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2003.08.012

DNA, nucleosomes or dead or dying cells [6 – 8]. Unlike other autoimmune diseases, SLE has therefore been studied primarily using spontaneous disease models [9], although an induced model would facilitate the investigation of the mechanisms and genetics of anti-DNA production. As now recognized, DNA has unique immunological properties that may affect its immunogenicity. Thus, depending on sequence and base methylation, DNA can be stimulatory, inhibitory or neutral with respect to mitogenic activity and cytokine induction [10 –17]. In particular, whereas bacterial DNA has broad stimulatory activities because of its content of CpG motifs [18], mammalian DNA fails to induce responses and, indeed, can inhibit responses to bacterial DNA and possibly other stimuli [15,19 –21]. Furthermore, bacterial DNA can induce a robust antibody response by immunization, while mammalian DNA lacks this ability [21,22]. The intrinsic inhibitory properties of

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mammalian DNA may result in poor immunogenicity and limit efforts to model lupus by immunization. Recently, we have investigated a new model for antiDNA production [23]. This model is based on the potency of synthetic oligonucleotides (ODN) as adjuvants [24,25]. These ODN contain an unmethylated CpG motif and have a phosphorothioate backbone to enhance activity. As shown with a variety of antigens, such ODN are highly effective adjuvants with activity similar to that of complete Freund’s adjuvant (CFA) [26 –28]. In our previous study, we showed that calf thymus (CT) DNA, complexed with methylated bovine serum albumin (mBSA), can induce antibodies to dsDNA when administered in incomplete Freund’s adjuvant (IFA) with a phosphorothioate CpG ODN [23]. Interestingly, immunization of DNA with CFA failed to elicit this response, suggesting particular effectiveness of CpG ODN as adjuvants for autoimmune responses. This effectiveness may derive from the ability of the ODN to counteract the inhibitory activity of mammalian DNA. In the current study, we have characterized further the anti-DNA antibodies induced in normal BALB/c mice by immunization with CT DNA with a CpG ODN as adjuvant. Specifically, we have addressed the isotype, cross-reactivity and avidity of these antibodies in comparison to anti-DNA antibodies arising spontaneously in autoimmune MRL/lpr mice. We also investigated other serological features of SLE in the sera of the immunized mice. In studies presented herein, we show that immunization with DNA with a CpG ODN induces anti-DNA antibodies that closely resemble those of spontaneous autoimmune mice in salient immunochemical properties. As such, these findings indicate that normal mice have the ability to generate potentially pathogenic specificities which may require the immunomodulatory effects of a CpG ODN for expression.

Materials and methods Animals Female BALB/c and MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mice were purchased from the Jackson Laboratory (Bar Harbor, NE, USA) and housed under conventional conditions in the animal facilities of Durham VAMC. Antigens DNA antigens were purchased from Sigma Chemical Co (St. Louis, MO, USA). Native calf thymus DNA (CT DNA) used for immunization was further purified by phenol-chloroform extraction and ethanol precipitation. To prepare dsDNA (dsDNA), the DNA was digested with S1 nuclease prior to purification. ss DNA was produced by boiling either native DNA or dsDNA for 10 min followed by rapid immersion in ice. The concentration of DNA was determined by OD260 readings with purity assessed by the OD260/

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OD280 ratio. Preparations in these experiments had a ratio of 1.7–1.9. DNA antigens from human placenta (HP), Escherichia coli (EC), Micrococcus lysodeikticus (MC), and Clostridium perfringens (CL) were similarly prepared. The phosphorothioate ODN used as an adjuvant had the sequence (5⬘-TCC ATG ACG TTC CTG ACG TT-3⬘) and was purchased from Midland Certified Reagents. Nucleosomes were made from fresh rat livers according to a standard protocol [29]. The preparation contained mainly mononucleosomes as determined by DNA size by agarose gel electrophoresis; the DNA in these preparations was predominantly in the range of 140 –160 bases. The nucleosome concentration was determined by OD260 value as for DNA. Calf thymus histones were purchased from Roche Diagnostics Corporation (Indianapolis, IN, USA). Immunization Female BALB/c mice were immunized with CT DNA complexed with mBSA and mixed with a CpG ODN in incomplete Freund’s adjuvant (IFA). Each mouse received 0.3 ml of emulsion containing 50 ␮g of CT DNA, 75 ␮g of mBSA, 10 ␮g of CpG oligonuleotide in IFA. Control groups received either CpG ODN alone, CT DNA with IFA, IFA alone, or nothing. Each group had 6 – 8 mice. Two boosters were given at 2 week intervals. Three weeks after the final boost, mice were bled from the retro-orbital sinuses under methoxyflurane anesthesia and sera obtained after clotting and centrifugation. Sera from 6 – 8 non-immunized MRL/lpr mice were used as positive controls. MRL/lpr mice were 4 –5 months of age at the time of bleeding. ELISA Anti-DNA activity was assessed by ELISA using, as antigen, photobiotinylated dsDNA added to Streptavidin coated plates [30]. For this purpose, the dsDNA was first biotinylated according to the manufacturer’s instruction (Vector Laboratory INC, Burlingame, CA, USA). Immulon 2HB microtiter plates (ThermoLab System, Franklin, MA, USA) were coated with Streptavidin (Sigma Chemical Co) at 5 ␮g/ml in 0.1M phosphate buffer, pH 9.0, 100 ␮l/well, and incubated overnight at 4°C. After three washes with PBS, biotinylated DNA at 0.2 ␮g/ml was added to wells and incubated at room temperature for 1 h. Sera diluted 1:100 in PBS-Tween (PBS containing 0.5% of Tween20) were added in duplicate and incubated for 1 h. Then 100 ␮l of peroxidase conjugated goat anti-mouse IgG (heavy and light chains, Sigma Chemical Co) diluted 1:1000 in PBS-Tween was added, and after 1 h incubation, followed by 200 ␮l of the substrate containing 0.015% of 3,3⬘,5,5⬘ tetramethylbenzidine (TMB) (Sigma Chemical Co.) in 0.1 M citrate buffer, pH 4.0, and 0.01% hydrogen peroxide for color development. After 35 min incubation, OD380 was determined with a microtiter plate reader (Molecular Dynamics, Menlo Park, CA, USA).

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For assessing anti-DNA isotypes, dilutions of the subclass specific secondary antibodies were first determined by titration. Briefly, Immulon 2HB microtiter plates were coated for 1 h at room temperature with purified IgG1, IgG2a, IgG2b, and IgG3 (Sigma Chemical Co.) made to 0.1 ␮g/ml in 0.1 M sodium bicarbonate buffer pH 9.6, then blocked with 0.2% bovine serum albumin for 1 h. Plates were washed three times with PBS. Two-fold dilutions of peroxidase conjugated HRP goat anti-mouse Ig subclass antibodies (Southern Biotechnology Associates, Inc.) were added to duplicates well on plates. TMB/citrate buffer/ hydrogen peroxide substrate was then added and plates were read at OD380 after 35 min incubation. The dilution producing the same OD result with its own subclass standard was chosen for the final isotyping assay which was performed in the same way as in the anti-DNA assay. For the isotype assay, sera were incubated with DNA at a 1:400 dilution. To measure the anti-nucleosome and anti-histone activities, Immulon 2HB plates were coated with either nucleosomes or histone (1 ␮g/ml in PBS). Two-fold dilutions of sera in PBS-Tween starting at 1:100 were added to wells. Subsequent steps were the same described above. To assess the anti-DNA avidity by ELISA, diluted sera (titrated to produce an OD380 of 1 when uninhibited) were mixed with two-fold dilutions of the inhibiting DNA (CTdsDNA or CTssDNA) starting at 200 ␮g/ml. The mixtures were incubated overnight at 4°C before adding to the plates coated with biotinylated CTdsDNA. Conjugate and substrate were added as above. The percentage inhibition was calculated as OD(no inhibitor)-OD(with inhibitor)/ OD(no inhibitor). Immune complex assay To assess the amount of DNA in immune complexes (IC), a fluorometric assay was used, with DNA detected by the dye PicoGreen (Molecular Probes, Eugene, OR, USA) [31]. PicoGreen binds specifically to dsDNA and allows detection of dsDNA in pg-ng range. Two-fold dilutions of CT DNA in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) starting at 1000 ng/ml were prepared as standards. Twenty ␮l of each serum was mixed with 20 ␮l of saturated ammonium sulfate in a 96 well V bottom polypropylene microtiter plate (Greiner Bio-one, Germany), incubated at 4°C for 1 h followed by centrifugation at 3000 rpm for 10 min. The supernatants were discarded and the precipitates were washed three times with 200 ␮l/well of 50% ammonium sulfate. The precipitates were then dissolved in 200 ␮l of TE buffer. Samples were added to duplicate wells (50 ␮l/well) in a Costar black polystyrene flat bottom 96 well plate (Costar Corning Incorporated, Costar, NY, USA), and mixed with 50 ␮l of PicoGreen (Molecular Probes Inc, Eugene, OR, USA) dye diluted 1:200 in TE. The plate was immediately read in a TECAN/GENios fluorometric microtiter plate reader (Salzburg, Austria) at an excitation wave-

length of 485 nm and emission wavelength of 535 nm. The DNA concentrations derived from a standard curve were multiplied by the dilution factor of 10 to give the DNA concentrations of the immune complexes. Results are expressed in ng/ml. Surface plasmon resonance (BIAcore) In these experiments, avidity measurements were performed using Streptavidin coated sensor chips (SA chip, BIACORE AB, Uppsala, Sweden) onto which the sonicated, biotinylated CT dsDNA antigen was immobilized. IgG in the sera was purified using protein G Sepharose 4 Fast Flow beads (Pharmacia Biotech AB, Uppsala, Sweden). The concentrations of the purified IgG were determined using the standard Bio-Rad Bradford assay (Bio-Rad, Hercules, CA, USA). SPR biosensor measurements were determined on a BIAcore 3000 (BIAcore Inc., Uppsala, Sweden) instrument and data analysis was performed using BIAevaluation 3.0 software (BIAcore Inc.). In the initial experiments, various concentrations of biotinylated CT dsDNA were immobilized onto a SA sensor surface (BIAcore AB, Sweden) to produce readings of 100 –1200 RU. Non-specific or bulk responses were subtracted from an in-line blank reference surface. Binding of purified IgG samples was monitored in real-time at 25°C with a continuous flow of PBS (150 mM NaCl, 0.005% surfactant p20), pH 7.4 at 30 ␮l/min. Bound IgG was removed and the sensor surfaces were regenerated following each cycle of binding by single or duplicate 5–10 ␮l pulses of regeneration solution (10 mM NaOH). A series of different IgG concentrations (100, 50, 25, 12.5, 6.25, and 1 ␮g/ml) from each mouse was injected over all three surfaces at 5–30 ␮l/min for 3–7 min. Final data were obtained from the experiments performed on the 600 RU sensor chip surface with the IgG being injected at 30 ␮l/min for 7 min, followed by 7 min dissociation time and 25 sec of regeneration time. DNA and IgG dilutions were made in PBS (Bio-Whittaker, USA) and 10 mM NaOH was used as regeneration buffer. The binding kinetics of the DNA-antiDNA complex were evaluated using the BIA evaluation package analysis and the simple Langmuir model to obtain best fits for the sensorgrams and to derive the on rates (Kon), off rates (Koff) and the dissociation constants (Kd). Statistical methods The statistical significance of the results was evaluated using the Mann-Whitney U test. Student’s t test was used when appropriate.

Results The magnitude of induced responses were first assessed in various immunization groups, using sera of MRL/lpr

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Fig. 1. Anti-DNA response in immunized and control mice. Groups of 6 – 8 female BALB/c mice were immunized with CT DNA using a CpG ODN as adjuvant in incomplete Freund’s adjuvant (IFA ⫹ CpG ⫹ CT); with CT DNA alone in IFA (IFA ⫹ CT); with a CpG ODN alone in IFA (IFA ⫹ CpG); or with IFA alone (IFA). Age-matched unimmunized BALB/c mice and MRL/lpr mice were used as negative and positive controls respectively. Presented are the mean OD380 values and standard deviations for each group.

mice as controls. As results in Fig. 1 show, immunization with DNA with a CpG ODN with IFA led to a significant anti-DNA response compared to those in the other groups (IFA alone, CpG ODN alone, and IFA plus CT DNA) and in unimmunized BALB/c mice (p ⬍ 0.01). While the induced responses in the BALB/c mice were greater than those in the other BALB/c groups, they were nevertheless lower than those of MRL/lpr mice (p ⬍ 0.01). These results are similar to those previously reported and confirm the effectiveness of a CpG ODN as an adjuvant [23]. The isotype distribution of induced anti-DNA was next assessed using isotype specific ELISA assays. As shown in Fig. 2, the predominant IgG subclass in the immunized mice was IgG2a, with lower amounts of IgG1 and IgG2b present. Very little IgG3 anti-DNA was detected. In contrast, for the MRL/lpr anti-DNA, IgG2a and IgG3 showed the highest levels, with lower levels of IgG1 and IgG2b present. The difference between the IgG3 anti-DNA responses of immunized BALB/c and MRL/lpr mice was significant (p ⬍ 0.01). To evaluate further the specificity of the induced and spontaneous anti-DNA, their binding to DNA from five different species (calf thymus, human placenta, E.coli, Micrococcus and Clostridium) was assessed by ELISA. As data in Fig. 3 indicate, induced and spontaneous anti-DNA antibodies showed reactivity to all of the antigens in the panel although, for the induced antibodies, some differences were observed in antibody levels to the different DNA antigens. These findings indicate that immunization, like spontaneous autoimmunity, leads to the expression of antibodies binding to determinants present widely on DNA

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Fig. 2. IgG isotype subclass distribution of induced and spontaneous anti-DNA antibodies. Sera of immunized BALB/c (IFA ⫹ CpG ⫹ CT) and MRL/lpr mice were assayed for isotype-specific anti-DNA antibodies using ELISA. Unimmunized BALB/c mice were used as negative controls. Data presented are the mean OD380 values and standard deviations for 6 – 8 mice in each group.

rather than a determinant specific for the immunizing CT DNA. The cross-reactivity of spontaneous anti-DNA is a characteristic feature of these antibodies and suggests predominant interaction with a structure on the DNA backbone [14]. In spontaneous SLE, anti-DNA antibodies are produced in association with antibodies to nucleosomes as well as nucleosome components such as histones. This pattern of expression has suggested that the nucleosome is the driving antigen for anti-DNA production, with epitope spreading generating a broad array of responses [7]. To determine whether immunization with DNA leads to the production of antibodies to other nucleosome components, anti-nucleo-

Fig. 3. Cross-reactivity of induced and spontaneous anti-DNA antibodies. Sera from 6 – 8 immunized BALB/c (IFA ⫹ CpG ⫹ CT) or MRL/lpr mice were tested for binding to DNA from calf thymus (CT), human placenta (HP), Escherichia coli (EC), Clostridium (CL), and Micrococcus (MC) DNA. Unimmunized BALB/c mice were used as negative controls. Data presented are the mean OD380 values and standard deviations for 6 – 8 mice in each group.

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T.T. Tran et al. / Clinical Immunology 109 (2003) 278 –287 Table 1 DNA immune complexes in immunized BALB/c and control mice DNA concentration in immune complexes (ng/ml) MRL/lpr IFA ⫹ CpG ⫹ CT IFA ⫹ CpG IFA BALB/c

Fig. 4. Anti-nucleosome activities in the sera of immunized BALB/c groups and MRL/lpr mice. Unimmunized BALB/c mice were used as negative controls. The figure shows the mean OD380 values for 6 – 8 mice in each group.

some and anti-histone antibody levels were measured by ELISA. As data in Fig. 4 indicate, mice receiving IFA all showed measurable levels of antibodies to nucleosomes; while significantly lower than those in MRL/lpr mice (p ⬍ 0.05), they were significantly higher than those in nonimmunized mice (p ⬍ 0.01). The mice receiving DNA, however, did not differ from the other groups. Similar findings were seen for anti-histone antibodies (Fig. 5). Together, these findings indicate that IFA alone can induce a response to nucleosomal antigens and that immunization with DNA does not promote epitope spreading to these antigens. An important mechanism for the pathogenicity of antiDNA is the formation of DNA-anti-DNA immune complexes (IC). To assess the presence of DNA-anti-DNA im-

Fig. 5. Anti-histone activities in the sera of immunized BALB/c groups and MRL/lpr mice. Unimmunized BALB/c mice were used as negative controls. The figure shows the mean OD380 values for 6 – 8 mice in each group.

1059.5 ⫾ 976.6 412.3 ⫾ 35.5 504.3 ⫾ 179.6 673.3 ⫾ 198.8 316.2 ⫾ 251.7

Immune complexes containing DNA were measured using PicoGreen as described in Materials and Methods. The results presented here are the mean DNA concentrations in the immune complexes and the standard deviations for 6 – 8 mice in each group. Normal BALB/c mice were immunized with CT DNA using CpG ODN in incomplete Freund’s adjuvant (IFA ⫹ CpG ⫹ CT); CpG alone in IFA (IFA ⫹ CpG); or IFA (IFA). Unimmunized BALB/c and MRL/lpr mice were used as negative and positive controls respectively.

mune complexes, a fluorometric assay was used to measure DNA in ammonium sulfate precipitates [31]. As shown in Table 1, whereas MRL/lpr sera had significant levels of DNA in precipitates, the sera of mice immunized with DNA had levels no greater than those of unimmunized mice. These results indicate that, while DNA immunization elicits an anti-DNA response, other conditions for the generation of immune complexes may not be met in this model. To evaluate further the properties of induced anti-DNA, binding avidity was assessed. While the macromolecular structure of DNA limits precise measurement of avidity, this assessment is nevertheless important for assessing patterns of antigen selection as well as potential for pathogenicity. For this purpose, the binding of the induced and spontaneous anti-DNA antibodies was compared by two different

Fig. 6. Binding specificities of induced and spontaneous anti-DNA antibodies by inhibition ELISA. Single stranded (ss) and double stranded (ds) CT DNA were used as inhibitors. 1:100 sera dilutions from immunized BALB/c (IFA ⫹ CpG ⫹ CT) and MRL/lpr mice were mixed with different concentrations of DNA inhibitors as described prior to adding to microtiter plates coated with biotinylated CTdsDNA. The percentage of inhibition was calculated as: OD380(no inhibitor)-OD380(with inhibitor)/OD380(no inhibitor).

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assays. We first used inhibition ELISA, testing both ss and dsDNA as inhibitors since many anti-DNA antibodies bind both antigens. As shown in Fig. 6, ssDNA was a much more effective inhibitor of binding than dsDNA for both induced and spontaneous anti-DNA. Nevertheless, the profile of inhibition was similar for both induced and spontaneous antibodies, suggesting a similar array of avidities and specificities. Surface plasmon resonance was next employed to obtain a more quantitative assessment of the avidity of these responses. As shown in representative sensorgrams in Fig. 7, in contrast to IgG from normal BALB/c mice, IgG from immunized BALB/c mice and from MRL/lpr mice showed specific binding to CT DNA (Fig. 7A). Figures 7B and 7C show titration curves obtained when different IgG concentrations were injected over the sensor chip surface. As these data indicate, equilibrium or steady-state binding was observed only at low IgG concentrations (1–12 ␮g/ml). From these assays, the binding kinetics of the induced and the spontaneous anti-DNA antibodies were calculated and are tabulated in Table 2. As these data indicate, there was no statistically significant difference in the on rates, off rates and dissociation constants (Kd) between the induced and spontaneous antibodies (p ⬎ 0.05). Coupled with the inhibition ELISA results, these findings suggest that immunization induces a similar array of specificities as present in spontaneous disease, with both induced and spontaneous antibodies showing cross-reactive DNA binding with a similar range of avidities.

Discussion Results presented herein demonstrate that the anti-DNA antibodies induced in normal mice by immunization resemble spontaneously produced anti-DNA antibodies in important immunochemical properties. Thus, the induced antibodies displayed similar specificity and avidity as spontaneous anti-DNA, with surface plasmon resonance showing similar kinetics and binding parameters. Since the induced antiDNA were generated in BALB/c mice, a strain without a known predisposition to autoimmunity, these findings suggest that, under appropriate circumstances, even normal mice can express autoantibodies, presumably reflecting the presence of potential autoreactive B cell precursors in their repertoire. In contrast to the current findings, previous efforts to model lupus by immunization with DNA have met with limited success. Indeed, mammalian DNA has generally been considered to be non-immunogenic and unable to induce a specific response under usual immunization conditions including complexation with a protein carrier and administration in CFA [21,22]. The poor immunogenicity of mammalian DNA contrasts to that of bacterial DNA which can elicit a robust anti-DNA response in normal mice by immunization under conditions in which mammalian DNA

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is inactive. With bacterial DNA as the immunogen in normal mice, however, the induced antibodies bind specifically to bacterial DNA and do not cross-react with mammalian DNA [21]. These induced antibodies appear to target sites, most likely sequences, that are present only on bacterial DNA. As such, these antibodies resemble antibodies to a foreign protein in their specificity for an epitope on the immunizing antigens. Despite their reactivity with DNA, antibodies induced to bacterial DNA in normal mice are not autoantibodies. While immunization with bacterial DNA does not elicit anti-DNA autoantibodies, the immunization experiments suggested that bacterial DNA has properties that promote immune responsiveness. As now recognized, bacterial DNA displays potent immunostimulatory activity that includes activation of B cells, macrophages, dendritic cells as well as the downstream effects of induced cytokines on various immune cells populations such as T cells [10 –14,16 –18]. These properties reflect the presence of short sequences motifs in bacterial DNA that are denoted as CpG motifs. These motifs center on an unmethylated CpG dinucleotide and occur much more commonly in bacterial DNA than mammalian DNA. Because of their activity, these motifs can serve as adjuvants, with bacterial DNA containing a “built-in” adjuvant. Because of its adjuvant activity, bacterial DNA can induce anti-DNA responses in normal mice, albeit with selective binding to bacterial DNA. In view of these observations and accumulating evidence that CpG ODN are highly effective adjuvants, we explored the use of a CpG ODN as an adjuvant to generate antibodies to mammalian DNA in an immunization model with normal mice. Indeed, as we showed, immunization with mammalian DNA with a CpG ODN elicits antibodies to mammalian DNA under conditions in which immunization with CFA is ineffective [23]. These observations point to the potency of CpG ODN as adjuvants and the feasibility of studying anti-DNA responses in an induced model. The studies presented herein confirm our previous observations and demonstrate the similarity of induced and spontaneous anti-DNA in their immunochemical properties. In terms of both cross-reactivity and avidity, the induced antibodies closely resembled those present in sera of spontaneous autoimmune antibodies. These observations are of interest since anti-DNA autoantibodies have not been elicited in normal mice by immunization with either bacterial or mammalian DNA in CFA [21,22]. Since immunization of preautoimmune NZB/NZW mice with bacterial, but not mammalian, DNA with CFA can elicit anti-DNA autoantibodies [22], these findings suggest that a CpG ODN can induce immunoregulatory changes that may resemble those in spontaneous autoimmunity and allow a response to DNA immunization. The requirement for a CpG ODN adjuvant may reflect either an enhanced activity compared to CFA or a unique ability to counteract the inhibitory activity of mammalian DNA. In this regard, the induced and sponta-

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Fig. 7. Binding kinetics of induced and spontaneous anti-DNA antibodies by SPR. IgG from immunized BALB/c (IFA ⫹ CpG ⫹ CT) and MRL/lpr mice was purified as described in Material and methods. The specific binding of anti-DNA antibodies to CT DNA is shown in Fig. 7A. IgG from an unimmunized BALB/c mouse was used to show non-specific binding. Fig. 7B and 7C show the typical sensorgrams obtained for different IgG concentrations from sera of an immunized BALB/c mouse and a MRL/lpr mouse respectively.

T.T. Tran et al. / Clinical Immunology 109 (2003) 278 –287 Table 2 Binding parameters of the induced and the spontaneous anti-DNA antibodies

Immunized BALB/c 1 2 3 4 MRL/lpr 1 2 3 4

On-rate ka (M⫺1 s⫺1)

Off-rate kd (s⫺1)

Dissociation constant Kd

1.7 ⫻ 103 7.3 ⫻ 103 5.6 ⫻ 103 1.6 ⫻ 104 2.6 ⫻ 103 1.8 ⫻ 104 2.0 ⫻ 104 1.7 ⫻ 104

1.1 ⫻ 10⫺3 3.1 ⫻ 10⫺3 7.5 ⫻ 10⫺3 2.0 ⫻ 10⫺2 1.3 ⫻ 10⫺3 6.4 ⫻ 10⫺3 7.4 ⫻ 10⫺3 5.7 ⫻ 10⫺3

0.66 ⫻ 10⫺6 M 0.43 ⫻ 10⫺6 M 0.98 ⫻ 10⫺6 M 1.20 ⫻ 10⫺6 M 0.49 ⫻ 10⫺6 M 0.35 ⫻ 10⫺6 M 0.37 ⫻ 10⫺6 M 0.33 ⫻ 10⫺6 M

These values were derived using the BIA evaluation package to obtain the best fits for the sensorgrams. The data shows the on-rates, off-rates and dissociation constants for IgG preparations from 4 mice in each group.

neous anti-DNA show differences in the expression of IgG3 isotype, possibly reflecting differences between cytokines induced in these setting as reflected in class switching [32,33]. While the exact role of the CpG ODN requires further investigation, these studies provide important evidence that normal mice can generate anti-DNA antibodies and that immunization can replicate at least certain aspects of DNA antigen drive. The most decisive evidence for this point comes from assessment of avidity. The assessment of antiDNA avidity by ELISA is problematic for a number of reasons including the size of the DNA antigen; uncertainty in the number, size and location of antigenic epitopes; the requirement for monogamous or bivalent interaction; and technical limitations of inhibition binding assays [34]. In general, anti-DNA binding has been considered to be high avidity and therefore indicative of antigen selection [35– 37]. The advent of surface plasmon resonance has provided a new approach for assessing anti-DNA interactions and provides a more precise assessment of avidity [38 – 41]. Surprisingly, this technique has not been widely used to study anti-DNA autoantibodies, although the results presented herein clearly show its utility. As our data indicate, induced and spontaneous anti-DNA antibodies show similarity in binding patterns as well as calculated avidities and on and off rates. A previous study using surface plasmon resonance to characterize murine anti-DNA response involved monoclonal antibodies and synthetic oligonucleotides as antigens [41]. In contrast, our preparations were obtained from serum and therefore likely represent a heterogenous population. Since our antigens are natural DNA of higher molecular weight, a direct comparison with the previous study is not possible, although the affinities we measured appear lower than those of the monoclonal antibodies. The calculated affinity constants in our study thus provide an assessment for polyclonal antibody preparations that could vary depending on the relative proportions of the different antibody subpopulations as well as molecular size of the antigens.

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The similarity in the induced and spontaneous antibodies is of interest in view of the presumed differences in the structure of the immunizing antigens. For the induced model, the immunogen consisted of a complex of DNA with the protein carrier mBSA whereas, in spontaneous autoimmunity, the nucleosome has been considered to be the most likely immunogen [7]. The DNA-mBSA complexes result from charge-charge interactions that lead to aggregation; in contrast, the nucleosome has an ordered structure in which DNA encircles a core of histone proteins. Nevertheless, both induced and spontaneous antibodies seem to bind DNA with similar specificity and avidity. These findings could suggest that both immunogens (i.e., DNA-mBSA complexes and nucleosomes) have exposed DNA regions that can act as B cell epitopes. Alternatively, DNA may separate from proteins sufficiently so that responses are directed in both situations to the DNA itself, without strict dependence on the presence of a protein to create an antigenic structure. Despite similarities in anti-DNA, the induced and spontaneous sera differed in their response to nucleosomes and histones. Thus, the sera of the DNA-immunized BALB/c mice had levels of antibodies to nucleosomes and histones similar to other groups exposed to IFA. These levels were all higher than those of unimmunized with mice but significantly lower than those of MRL/lpr mice. These findings are of interest since they suggest that the production of antibodies to histones and other nucleosomal proteins is not necessarily linked to antibodies to DNA. They further suggest that administration of mineral oils present in IFA can promote at least certain autoantibody response, perhaps reflecting a mechanism similar to that for pristane [42– 44]. In longitudinal studies of autoimmune mice, there is evidence that anti-DNA antibodies can emerge during an ongoing response to nucleosomes, reflecting a type of epitope spreading [45,46]. In our studies, we did not observe a comparable pattern of spreading, since the induced antiDNA response did not lead to an anti-nucleosome or antihistone response. The inability to detect epitope spreading in the DNA-immunized mice may result from the time period of observation or the lack of sufficient endogenous nucleosomal antigen to stimulate B or T cell responses. Studies are in progress to follow immunized mice over time to see if any other autoantibody responses occur. Finally, while the induced and spontaneous anti-DNA antibodies showed similar properties, the serum of the immunized mice did not show DNA-containing immune complexes in a fluorometric assay. This assay uses the dye PicoGreen to detect DNA in complexes that precipitate with ammonium sulfate, with previous studies showing that this material consists of IgG-containing complexes [31]. The low levels of complexes in the immunized mice may result from a number of factors including the lack of available endogenous antigen to form complexes; the deposition of any complexes that form into the tissues; or the lack of “pathogenic” or ’nephritogenic” antibodies in the induced sera [47]. In this regard, we cannot exclude the possibility

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that the sera of immunized mice contain complexes that are not detected by ammonium sulfate precipitation but would be demonstrable by some other immunochemical approach. Nevertheless, given the advantages of an induced model for autoimmunity, our system will allow systemic exploration of the determinants of complex formation, deposition and clearance. In conclusion, this study extends previous observations on induced anti-DNA production and uses a variety of immunochemical approaches to demonstrate the similarity between induced and spontaneous anti-DNA response. Importantly, they confirm the unique properties of CpG ODN as adjuvants and their ability to enhance responses to both foreign and self antigens. Future studies will characterize the pathogenicity of induced antibodies and elucidate the cellular changes induced by CpG DNA that are keys to the development of auto-reactivity.

Acknowledgments This work was supported by a VA Merit Review grant and by NIH grant RO1 AI44808.

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