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(Journal of Leukocyte Biology. 2001;70:103-112.)
© 2001 by Society for Leukocyte Biology

Immunostimulatory CpG-modified plasmid DNA enhances IL-12, TNF-{alpha}, and NO production by bovine macrophages

Lisl K. M. Shoda*, Kimberly A. Kegerreis*, Carlos E. Suarez{dagger}, Waithaka Mwangi*, Donald P. Knowles* and Wendy C. Brown*

* Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, and
{dagger} Animal Disease Research Unit, United States Department of Agriculture, Agricultural Research Service, Washington State University, Pullman, Washington

Correspondence: Dr. Wendy C. Brown, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040. E-mail: wbrown{at}vetmed.wsu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The immunogenicity of DNA vaccines is partially attributable to the adjuvant properties of bacterial plasmid DNA (pDNA) for B lymphocytes and professional antigen-presenting cells. In mice, modification of immunostimulatory sequences (ISSs), including CpG motifs, in pDNA vectors or oligodeoxynucleotides can increase or decrease their adjuvant properties. ISSs that stimulate optimal responses reportedly differ for murine and human leukocytes. We have previously characterized the mitogenic properties of oligodeoxynucleotides containing one AACGTT motif for bovine B lymphocytes. We now define cytokine responses by macrophages stimulated with pDNA engineered to contain an ISS comprising two AACGTT motifs. Macrophages activated with CpG-modified pDNA secreted significantly more interleukin-12, tumor necrosis factor-{alpha}, and nitric oxide than macrophages stimulated with unmodified pDNA or modified pDNA that contained nucleotides scrambled to remove CpG motifs. Engineered CpG-pDNA or CpG-oligodeoxynucleotides should be useful as vaccines or adjuvants to promote the enhanced type 1 responses important for protection against intracellular pathogens.

Key Words: immunostimulatory DNA sequences (ISSs) • DNA vaccines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Microbial DNA, historically considered an immunologically inert material, is now known to stimulate antigen-presenting cells to direct a T helper (Th)1 response [1 2 3 4 ]. The immunostimulatory sequences (ISSs) in bacterial and other nonvertebrate DNAs, defined as CpG motifs, were originally identified as hexamers composed of a central CG dinucleotide flanked by two 5' purines and two 3' pyrimidines [5 ]. It is now appreciated that immunostimulatory CpG motifs are far more diverse than previously believed [4 ]. Methylation of the cytosine in the CG dinucleotide abolished mitogenic activity, indicating that the presence of unmethylated CG dinucleotides is critical for recognition [6 ]. In bacterial DNA, the CG dinucleotide content approximates the expected frequency of 0.0625 dinucleotide pair, and the cytosine moieties are generally unmethylated. By contrast, mammalian DNA contains fewer CG dinucleotides than the predicted value, and the cytosine moieties are generally methylated [1 4 ].

Immune-cell activation induced by CpG motifs has been extensively studied in mice. Bacterial DNA or CpG oligodeoxynucleotides (ODNs) stimulate dendritic cells and macrophages to secrete cytokines including interleukin (IL)-12, tumor necrosis factor (TNF)-{alpha}, and IL-18 [7 8 9 10 ] and to increase expression of the costimulatory molecules major histocompatibility complex class II, CD80, and CD86 [11 ]. Furthermore, the enhanced production of interferon (IFN)-{gamma} by CpG DNA-activated natural killer cells is mediated by IL-12 [12 ]. CpG DNA also induces the production of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in IFN-{gamma}-primed murine macrophages [13 ], and in the absence of IFN-{gamma}, suboptimal amounts of lipopolysaccharide (LPS) can synergize with CpG DNA to induce iNOS and NO in a macrophage cell line [14 ]. Bacterial DNA and CpG ODNs are also mitogenic for B cells and enhance immunoglobulin (Ig)M and IgG production [6 , 8 , 15 ]. Thus, the identification of CpG motifs has provided a means of promoting the IFN-{gamma}-dominant Th1 immunity that is important for protection against intracellular pathogens.

The adjuvant property of CpG motifs has provided one explanation for the exceptional immunogenicity of DNA vaccines in mice, which is characterized by stimulation of cytotoxic T lymphocytes, high levels of IFN-{gamma}, a dominant IgG2a response, and protective immunity against intracellular pathogens and viruses [reviewed in ref. 16 ]. DNA vaccines are composed of plasmid DNA (pDNA) encoding the antigen of interest but also contain CpG motifs within the noncoding portion of the pDNA. Methylation of these CpG motifs abrogated the Th1-inducing properties of a DNA vaccine [17 ]. Furthermore, by the addition of CpG motifs, pDNAs could be engineered to improve their adjuvanticity for mice [18 19 20 ]. Thus, CpG-containing pDNA provides the dual functions of delivering antigen and serving as a Th1 adjuvant.

Fewer studies have examined the adjuvant effects of bacterial DNA for nonrodent species. Genomic Escherichia coli DNA and certain CpG ODNs stimulated human B-cell proliferation and production of inflammatory cytokines, including IL-6, IL-12, IL-18, and TNF-{alpha}, by peripheral blood mononuclear cell (PBMC)-derived monocytes and dendritic cells [18 , 21 22 23 24 ]. One group of researchers has reported the induction of type I IFN, IL-12 p40, and IL-18 by human PBMCs transfected with pDNA [18 ]. In general, CpG motifs that maximally induce human leukocyte responses differ from those reported for murine leukocytes [4 ]. The adjuvant properties of CpG oligonucleotides have been less-well characterized in ruminants. E. coli DNA stimulated bovine B-cell proliferation and IgG secretion, and immunostimulatory AACGTT and GACGTT motifs were identified [25 ]. We recently demonstrated that E. coli DNA induced secretion of IL-12, TNF-{alpha} and NO by macrophages [26 ]. However, additional information on immune modulation by CpG sequences stimulatory for nonrodent species is clearly needed to optimize the development of nucleic acid vaccines for infectious diseases pertinent to a specific host.

DNA vaccines against viral and protozoan pathogens have been employed in cattle [27 28 29 30 31 32 ] with limited success. In most studies, the mechanism of protection was not determined. However, in two studies, protective immunity against bovine herpesvirus was associated with increased IgG2 and IFN-{gamma} production [30 , 32 ]. Immunization of cattle with a pDNA vaccine encoding major surface protein-1a of the ehrlichial pathogen Anaplasma marginale resulted in detectable antigen-specific T-cell-proliferative, IFN-{gamma}, and antibody responses [33 ]. However, these responses were relatively weak, and the antibody response was limited to IgG1, suggesting that insufficient IFN-{gamma} was produced to promote isotype switching [34 ]. Because type 1 immune responses, characterized by elevated IgG2 and IFN-{gamma} production, are associated with protection against A. marginale [35 ] as well as the hemoprotozoan parasite Babesia bovis [36 ], the present studies were undertaken to improve the adjuvant activity of pDNA vectors for use in immunization against these bovine pathogens. We demonstrate the activation of bovine macrophages by pDNA and show that inclusion of additional AACGTT motifs within a pDNA vector enhances production of inflammatory cytokines and NO by bovine monocyte-derived macrophages. Such engineered vectors should be useful for immunization against A. marginale and other intracellular pathogens of cattle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oligodeoxynucleotides
The phosphodiester ODNs designated CS ODN, CpG-methylated CS (mCS ODN), and CS-scrambled ODN (CSS ODN) were synthesized by Oligos Etc. (Wilsonville, OR). The ODN sequences are shown in Table 1 . CSS ODN contains the same nucleotides as CS ODN, but they are scrambled to eliminate the AACGTT motifs. Lyophilized aliquots of ODNs were stored at -80°C.


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  		Table 1. Oligodeoxynucleotides Used in This Study

 
Plasmid DNA
The VR1055 vector (Vical Inc., San Diego, CA) was modified to include the CpG CS ODN, 5'-GAACGTTGGATCCAACGTT-3'. To clone CS ODN into the VR1055 vector, the complementary ODNs CS-F (5'-GAACGTTGG ATCCAACGTTG-3') and CS-R (5'-GATCCAACGTTGGATCCAACGTTCTGCA-3'), each containing a 5' PstI restriction site and a 3' BamHI restriction site, were designed. To generate double-stranded DNA ODNs, 50 µg of single-stranded CS-F and CS-R were incubated at 37°C for 2 h; this was followed by ethanol precipitation, drying, and reconstitution in 50 µL of deionized water. The vector VR1055 was digested with PstI and BamHI restriction enzymes, and the double-stranded CS ODN was ligated overnight into the VR1055 multiple cloning site at 14°C. The ligation product, termed VR1055-CS, was used to transform competent E. coli and plated to Luria-Bertani–kanamycin plates. The colonies were analyzed using PCR primers Vical-F1 (5'-GGCTATATGCCAATACTCTG-3'), representing a sequence located ~600 bp upstream of the polylinker site, and CS-R. Plasmids containing the CS ODN produced 600-bp bands when subjected to agarose electrophoresis. The unmodified VR1055 vector was used as a negative control. Positive clones were sequenced using primer Vical-F2 (5'-CAGATGGAAGACTTAAGGCAGC-3'). Sequencing verified that clones contained the ODN sequence in the correct orientation. Essentially endotoxin-free pDNA was purified using an EndoFree Plasmid Maxi kit in accordance with the manufacturer’s (Qiagen, Valencia, CA) instructions. For some experiments, pDNA was linearized by digestion with KpnI. Unmodified VR1055 or VR1055 ligated with a scrambled version of CS, 5'-GAACTTGGGATCCACAGTTG-3', termed VR1055-CSS, served as negative controls in cell culture experiments. These were also cloned into the PstI and BamHI sites in the VR1055 pDNA.

Limulus amoebocyte lysate assay
Cell culture reagents, pDNA, and ODNs were tested for trace amounts of endotoxin by using the Limulus amoebocyte lysate assay according to the manufacturer’s (BioWhittaker, Inc., Walkersville, MD) instructions. All pDNA (25 µg/mL) and ODN (10 µM) samples contained <0.06 endotoxin units (EU)/mL (<6 pg/mL) of endotoxin, which is the limit of sensitivity of the assay. A 25-µg/mL solution of E. coli DNA contained 0.06 EU/mL of endotoxin.

Monoclonal antibodies
Unless indicated otherwise, monoclonal antibodies (mAbs) were purchased from the Washington State University Monoclonal Antibody Center, Pullman.

B-lymphocyte isolation
B lymphocytes were isolated from bovine PBMCs by positive selection as previously described [25 ]. Briefly, PBMCs were resuspended at a concentration of 107/mL with 3 µg/mL of bovine CD21-specific mAb GB25A in complete medium, consisting of RPMI-1640 (Gibco-BRL, Rockville, MD) supplemented with 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; Gibco-BRL), 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine (Mediatech, Inc., Herndon, Va.), 50 µg/mL of gentamicin sulfate (Gibco-BRL), and 5 x 10-5 M 2 mercaptoethanol (Sigma Chemical Co., St. Louis, MO). The cell suspension was incubated at 4°C for 40 min with gentle agitation, and bead-bound B cells were isolated using goat anti-mouse IgG-coated magnetic beads (Dynabead M-450; Dynal Inc., Lake Success, NY) in accordance with the manufacturer’s instructions. The procedure routinely yielded >90% surface-Ig-expressing cells. The purified cell population was negative for cells expressing CD14, CD2, CD3, CD4, CD8, or the {gamma}/{delta} T-cell receptor, as determined by fluorescence-activated cell sorting analysis using mAbs specific for bovine CD14 (CAM36A), CD2 (mAb MUC2A), CD3 (mAb MM1A), CD4 (mAb CACT 138A), CD8 (mAbs CACT 80C and BAT 82B), and the {delta} chain of {gamma}/{delta} T-cell receptor (mAb CACT 61A).

B-cell proliferation assays
Triplicate cultures of purified B cells (2 x106/mL) in 100-µL volumes of complete RPMI 1640 medium containing 1 µg/mL of pokeweed mitogen (Sigma), 1 µg/mL of concanavalin A (Sigma), linearized pDNA (3.75–25.0 µg/mL), or ODN (4.375–35.0 µM) were incubated for 72 h at 37°C. Polymyxin B sulfate (final concentration, 10 µg/mL) was included in cultures stimulated with pDNA. For the last 18 h of culture, B cells were radiolabeled with 0.25 µCi of [3H]uridine (New England Nuclear, Boston, MA). The cells were harvested, and radioactivity was determined in a liquid scintillation counter. Results are presented as mean counts per minute of replicate cultures ± 1 SD. Proliferation was analyzed for statistical significance by the Mann-Whitney test.

Macrophage isolation and culture
Monocyte-derived macrophages were isolated from PBMCs of six adult cattle by plastic adherence and culturing for 6–7 days in complete RPMI 1640 medium [26 , 37 , 38 ]. After culture, adherent macrophages were harvested with Ca2+- and Mg2+-free Hanks balanced salt solution containing 0.5 mM EDTA. The procedure regularly yielded >80% CD14-expressing cells, as determined by fluorescence-activated cell sorting analysis and staining with mAb CAM36A. Macrophages were cultured for 6 h (for RNA isolation) or 24–48 h (for supernatant collection) with medium or with 25 µg/mL of unmodified VR1055 or modified VR1055-CS or VR1055 CSS pDNA in the presence of 50 U/mL of bovine recombinant IFN-{gamma} [Ciba-Geigy; kindly provided by Lorne Babiuk, Veterinary Infectious Disease Organization (VIDO), Saskatoon, Saskatchewan, Canada] plus 10 µg/mL of polymyxin B sulfate (Sigma) in 24-well plates at a density of 5 x 105 cells per well in 0.5-mL volumes. As positive controls, macrophages were treated with 25 µg/mL of E. coli DNA or 100 ng/mL of LPS from E. coli (O55:B5; Sigma) plus IFN-{gamma}. In some experiments, macrophages were treated with 10 µM ODN in complex with 0.05 mg/mL of Lipofectin (Gibco-BRL) according to the manufacturer’s instructions. Although Lipofectin is not required for the activation of bovine macrophages by bacterial or protozoan DNA [26 ] or for activation of bovine B cells [25 ] or monocytes by CpG ODN, we have repeatedly observed that the presence of Lipofectin is required for activation of bovine macrophages by CpG ODN at all concentrations tested (0.2–25 µM) [Y. Zhang, W. C. Brown, unpublished results].

Reverse transcription-PCR
The induction of cytokine and iNOS mRNA production by macrophages was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) as previously described [26 , 38 ]. RNA was isolated by using TRIzol reagent (Gibco BRL) and treated with deoxyribonuclease (Ambion, Inc., Austin, TX), and PCR amplification was conducted with gene-specific primers. The primers for bovine IL-12 p40, IL-12 p35, TNF-{alpha}, IL-1ß, iNOS, and ß-actin and the conditions used for RT-PCR were described recently [26 , 38 ]. The cycle number chosen for each primer set was empirically determined for each set of samples on the basis of the positive control and was selected to fall within the linear range of amplification. Sample target signals were normalized to their corresponding ß-actin signals, and the normalized values were compared.

Detection of IL-12 by dot blot assay
Supernatants from macrophages treated with ODN or pDNA were analyzed for the presence of IL-12 by using mAb 17827, which is specific for bovine IL-12 p40 (Serotec, Raleigh, NC), as described previously [26 ]. Macrophages were cultured in serum-free Iscove’s medium supplemented with 2 mM L-glutamine (Mediatech, Herndon, VA), 25 mM HEPES, 50 µg/mL of gentamicin sulfate, and 5 x 10-5 M 2-mercaptoethanol (Sigma). Supernatants and recombinant human IL-12 (kindly provided by Genetics Institute, Inc., Cambridge, MA) were serially diluted, and 200 µL were applied to a nitrocellulose membrane by using a HybriDot vacuum filtration manifold (Gibco BRL). Bound IL-12 was identified using the Western Star chemiluminescence detection system (Tropix, Inc., Bedford, MA) essentially as described by the manufacturer. Briefly, the membrane was incubated on a rocker for 1 h at room temperature or overnight at 4°C with I-block blocking solution (part of the Western Star kit; Tropix) followed by incubation for 1 h at room temperature or overnight at 4°C with IL-12 p40-specific mAb at a final concentration of 1 µg/mL in I-block. After six 15-min washes with TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.6), alkaline phosphatase-conjugated goat anti-mouse IgG + IgM diluted 1:15,000 in I-block was added for 1 h. The membrane was washed as described above, incubated twice for 2 min in 1x assay buffer (20 mM Tris, pH 9.8;1 mM MgCl2), and transferred to a Western Star development folder. The substrate solution, composed of 3 mL of CDPStar-RTU and 150 µL of NitroBlock (included in the Western Star kit), was spread evenly over the membrane and allowed to bind for 5 min. Excess solution was eliminated by smoothing out the development folder, and the blot was exposed to autoradiography film.

IL-12 bioassay
To confirm that the IL-12 detected by dot blot assay was biologically active, a bioassay was conducted with macrophage supernatants. The bioassay is based on the ability of IL-12 to induce IFN-{gamma} production by PBMCs [26 , 38 ]. Briefly, 200 µL of macrophage culture supernatant or, to create a standard curve, 0.1–1,000 pg/mL of recombinant human IL-12 were incubated for 2 days with 2 x 106 PBMCs and 1 µg/mL of phytohemagglutinin in a total volume of 400 µL/well in a 48-well plate. Supernatants from stimulated PBMCs were collected and assayed in duplicate for IFN-{gamma} by using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s (CSL Limited; Parkeville, Victoria, Australia) instructions. IFN-{gamma} activity was determined from a standard curve derived with a T-cell supernatant estimated, by the vesicular stomatitis virus cytopathic-effect reduction assay, to contain 440 U/mL of IFN-{gamma}. Macrophage supernatants were also evaluated for residual exogenous IFN-{gamma}, because some macrophage cultures contained 50 U/mL of recombinant bovine IFN-{gamma}. Significant differences in IFN-{gamma} production were determined by the one-tailed Student t-test.

Detection of nitrite by the Griess assay
Nitrite (NO2-) present in macrophage culture supernatants was measured in a Griess assay [26 , 38 ]. Briefly, macrophages were cultured for 48 h at a density of 0.5 x 105–1 x 105 cells/well with 25 µg/mL of DNA, 50 U/mL of IFN-{gamma}, and 10 µg/mL of polymyxin B sulfate in 96-well flat-bottomed plates. Culture supernatants were transferred (50 µL/well) to new 96-well, flat-bottomed plates, 50 µL/well each of 1% (w/v) sulfanilamide (Sigma) in 2.5% H3PO4 and, subsequently, 0.1% (w/v) naphthylethylenediamine dihydrochloride (Sigma) in 2.5% H3PO4 were added to the supernatants, and the absorbance at 540 nm was compared to an NaNO2 standard curve. Results are presented as the mean micromolar concentrations of NO2- in triplicate cultures ± 1 SD. Accumulation of NO2- was analyzed for statistical significance by the one-tailed Student t-test.

Detection of TNF-{alpha} by ELISA
Macrophages were cultured for 24 h with medium, pDNA, genomic E. coli DNA (positive control), or genomic bovine (Bos taurus) DNA (negative control) plus 50 U/mL of IFN-{gamma} and 10 µg/mL of polymyxin B sulfate. Supernatants were serially diluted and analyzed for TNF-{alpha} by ELISA [26 , 38 , 39 ]. Immulon II ELISA plates (Dynax Technologies, Chantilly, VA) were coated with anti-bovine TNF-{alpha} mAb 1D11-13 (kindly provided by Dale Godson, VIDO). Plates were washed six times with TBST. Samples diluted in TBST containing 0.5% gelatin were added to the plates and incubated for 2 h at room temperature or overnight at 4°C. Bound TNF was detected by incubation with a rabbit anti-TNF-{alpha} serum (VIDO) and, subsequently, with biotinylated goat-anti rabbit IgG (heavy and light chains; Zymed Laboratories, San Francisco, CA), streptavidin-alkaline phosphatase (Gibco BRL), and the substrate p-nitrophenyl phosphate [di(Tris) salt; crystalline]. The reaction was stopped by addition of 0.3 M EDTA (pH 8.0), and the optical density at 405 nm was determined with an ELISA plate reader. Samples were analyzed against recombinant bovine TNF-{alpha} (VIDO) diluted to 0.02–10.0 ng/mL as a standard. Statistical analysis of TNF-{alpha} levels was performed with the one-tailed Student t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Determination of endotoxin sensitivity of bovine macrophages
The levels of endotoxin in our reagents were undetectable or very low (the E. coli DNA preparation contained 6 pg/mL). However, because human monocyte-derived macrophages are sensitive to 100-fold-lower levels of endotoxin than are murine macrophages [23 ], it was important to determine the sensitivity of bovine macrophages to endotoxin and rule out the possibility of trace amounts of endotoxin in our samples affecting the results. LPS at <10 ng/mL failed to induce mRNA transcription for IL-12 p40, TNF-{alpha}, and IL-1ß (Fig. 1A ). Induction of iNOS was more sensitive to endotoxin; iNOS mRNA was detectable using >=1 pg/mL of LPS. However, inclusion of 10 µg/mL of polymyxin B sulfate was able to abrogate the effects of <=10 ng/mL of LPS on cytokine and iNOS transcription.



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  		Figure 1. Macrophage responses to serially diluted LPS. (A) RNA collected from macrophages incubated for 6 h with serially diluted LPS (0.001–10 ng/mL) in the absence or presence of 10 µg/mL of polymyxin B sulfate was analyzed for the indicated cytokines, iNOS, or ß-actin by RT-PCR. (B) Supernatants collected from macrophages cultured in triplicate for 48 h with serially diluted LPS (0.001–100 ng/mL) and 50 U/mL of IFN-{gamma} in the absence (white bars) or presence (black bars) of 10 µg/mL of polymyxin B sulfate were analyzed by the Griess assay. These data are representative of four independent experiments.

 
Since iNOS mRNA was the most sensitive indicator of LPS stimulation, NO production was also used to determine the limit of LPS sensitivity of bovine macrophages. In the absence of polymyxin B sulfate, detectable NO2- was induced by >=0.01 ng/mL of LPS (Fig. 1B) . However, the addition of polymyxin B sulfate blocked NO2- production when <=1 ng/mL of LPS was used. Because all of our samples contained <=0.006 ng/mL of endotoxin and all cultures were performed in the presence of polymyxin B sulfate, we ruled out the possibility that endotoxin was responsible for the results obtained with DNA or ODN.

Activation of B cells and macrophages by CpG ODNs containing the AACGTT motif
An ODN containing two AACGTT motifs (CS ODN) and control ODNs containing the same sequence with methylated cytosines in the CG base pairs (mCS ODN) or containing a scrambled sequence (CSS ODN) were constructed to verify the CpG-dependent mitogenic activity of the CS ODN sequence toward B cells. The AACGTT CpG motif was selected because it was known to stimulate bovine B-cell proliferation [25 ]. As observed previously with a different ODN, containing a single AACGTT motif [25 ], CS ODN was strongly mitogenic for B cells, whereas neither mCS ODN nor CSS ODN was mitogenic (Fig. 2A ). A preliminary experiment was also conducted to determine if this ODN sequence stimulated macrophages. Macrophages incubated with CS ODN, but not with mCS ODN, in the presence of Lipofectin produced IL-12 p40 (Fig. 2B) . These data show that ODNs containing two unmethylated CpG AACGTT motifs activate both B cells and macrophages of cattle.



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  		Figure 2. Stimulation of CpG-dependent B-cell proliferation and macrophage IL-12 p40 production by CS ODN. (A) B cells were cultured in triplicate wells for 72 h with 5–40 µM CS ODN (open squares), CSS ODN (closed circles), or mCS ODN (closed diamonds). Results are mean counts per minute ± 1 SD of data from triplicate cultures. The level of proliferation is significantly higher than that induced by medium, CSS ODN, or mCS ODN (P <0.05, indicated by an asterisk). These data are representative of three independent experiments. (B) The upper blot contains serial dilutions (0.4–12.5 ng) of human IL-12. The lower blot contains serial dilutions (1:2–1:128) of supernatants from macrophages treated with Lipofectin alone, CS ODN plus Lipofectin, mCS ODN plus Lipofectin, or E. coli DNA. IL-12 was identified after incubation of the blots with a mAb specific for bovine IL-12 p40 and visualized using an alkaline phosphatase conjugate detection system. Autoradiograph films are shown.

 
pDNA induces transcription of macrophage cytokine and iNOS mRNAs which is enhanced by addition of CpG motifs
ISSs containing CpG motifs induced the production of IL-12 and TNF-{alpha} by murine macrophages and human monocytes [12 , 22 , 23 ]. For mice, the original stimulatory CpG motifs that were described consisted of a central CG dinucleotide flanked by 5'-purines and 3'-pyrimidines and included GACGTC, AGCGCT, AACGTT, AACGCT, and AACGTC. However, it has more recently been determined that a broader repertoire of CpG motifs is capable of leukocyte activation [40 , 41 ]. We selected the VR1055 pDNA vector to characterize CpG activity toward bovine macrophages. Unmodified VR1055 pDNA contains seven classical CpG motifs, five in the format GACGTC, one GACGTT, and one AACGCT. Our earlier studies showed that the AACGTT and GACGTT motifs stimulated bovine B cells [25 ], and the 2059 ODN, which contains three copies of the GTCGTT motif and activates human PBMCs and B cells [23 , 41 ], is also mitogenic for bovine B cells [Y. Zhang, W. C. Brown, unpublished observations]. Although additional nonclassical immunostimulatory CpG motifs may be present in the VR1055 pDNA, their putative immunostimulatory properties for bovine leukocytes have not been demonstrated. Therefore, to investigate the possibility of amplifying macrophage responses to pDNA through the inclusion of additional CpG motifs, we selected the immunostimulatory AACGTT motif, which is known to activate bovine B cells but is not present in VR1055 pDNA, for use in engineering the pDNA.

VR1055 pDNA was modified to include the sequence present in the immunostimulatory CS ODN containing two AACGTT CpG motifs (VR1055-CS). Experiments measuring levels of cytokine and iNOS transcripts in pDNA-stimulated macrophages were performed prior to measurement of secreted protein. Incubation of macrophages with unmodified VR1055 pDNA demonstrated increased transcription of the genes encoding the IL-12 p40 and p35 subunits, TNF-{alpha}, IL-1ß, and iNOS compared with untreated macrophages (Fig. 3 ). In cells stimulated with modified VR1055-CS pDNA, transcription of the IL-12 p35 subunit, IL-1ß, and iNOS genes was further enhanced by approximately eight-, three-, and threefold, respectively, over that induced by unmodified VR1055 pDNA (Fig. 3C 3E and 3F) . VR1055-CS pDNA moderately enhanced production of IL-12 p40 (Fig. 3B) and did not measurably up-regulate TNF-{alpha} transcription beyond that induced by unmodified pDNA (Fig. 3D) . These results are representative of three experiments performed with macrophages from different cattle and two different pDNA preparations. In one experiment, VR1055-CSS pDNA was assayed and found to have stimulatory activity comparable with or less than that of unmodified VR1055 pDNA (data not shown).



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  		Figure 3. Induction of cytokine and iNOS mRNA transcription by macrophages treated with unmodified and CpG-modified pDNA. Macrophages were cultured for 6 h with 25 µg/mL of pDNA, 50 U/mL of IFN-{gamma}, and 10 µg/mL of polymyxin B sulfate or 1 µg/mL of LPS plus IFN-{gamma}. RNA was isolated, treated with DNase, and analyzed by RT-PCR for expression of the indicated cytokines, iNOS, and ß-actin. (A) Representative RT-PCR products for the indicated cytokines are shown for one experiment in which macrophages were treated with medium alone, LPS, E. coli DNA, VR1055 pDNA, and VR1055-CS pDNA. The relative amounts of IL-12 p40 (B), IL-12 p35 (C), TNF-{alpha} (D), IL-1ß (E), and iNOS (F) transcript induced by the indicated treatments are shown. These data are representative of three independent experiments.

 
Modified VR1055-CS pDNA stimulates higher-level IL-12, TNF-{alpha}, and NO protein production than unmodified VR1055 pDNA
Assays measuring both IL-12 p40/p70 heterodimer production and IL-12 activity were performed. In four experiments using two different preparations of pDNA, macrophages treated with modified VR1055-CS pDNA secreted approximately two- to fourfold more IL-12 p40 than macrophages treated with unmodified VR1055 pDNA (Fig. 4 ). Control VR1055-CSS pDNA did not stimulate IL-12 p40 production to a level above that of unmodified VR1055 pDNA. This assay does not distinguish heterodimeric IL-12 p70 from IL-12 p40 protein. However, these data, combined with the increase in IL-12 p35 and p40 transcription (Fig. 3B and 3C) , suggested that production of the IL-12 heterodimer was enhanced by the inclusion of two AACGTT motifs in VR1055 pDNA.



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  		Figure 4. Modified VR1055-CS pDNA induces IL-12 p40 secretion. The upper blot contains serial dilutions (0.4–12.5 ng) of human IL-12. The lower blot contain serial dilutions (1:2–1:128) of supernatants from macrophages cultured with the following: serum-free medium, unmodified VR1055 pDNA, control ODN-modified VR1055-CSS pDNA, CpG-modified VR1055-CS pDNA, and E. coli DNA. IL-12 was identified by incubating the blots with a mAb specific for bovine IL-12 p40 and visualized using an alkaline phosphatase conjugate detection system. Autoradiograph films are shown. These data are representative of four independent experiments.

 
Antibody reagents for measurement of bovine IL-12 p35 and p70 are not available, so a bioassay was performed to provide additional evidence for production of the functional IL-12 heterodimer. The assay is based on the ability of IL-12 to induce production of IFN-{gamma} by bovine PBMCs [26 , 38 ]. All macrophage supernatants contained <10 U/mL of IFN-{gamma}. Supernatants from macrophage cultures treated with unmodified VR1055 pDNA induced significantly more IFN-{gamma} production than did those from unstimulated macrophages, and macrophages cultured with modified VR1055-CS pDNA induced significantly more IFN-{gamma} production than the unmodified VR1055 pDNA (P <=0.003) (Fig. 5A ). In contrast, in this experiment, macrophages treated with bovine DNA or VR1055-CSS pDNA containing sequences scrambled to remove the CpG motifs did not stimulate IFN-{gamma} production above background levels. By comparison with the standard curve for human IL-12, it was determined that the VR1055 supernatant contained the equivalent of 16.5 ± 0.2 pg/mL of IL-12 and that the VR1055-CS supernatant contained the equivalent of 38.9 ± 0.6 pg/mL of IL-12 (a 2.4-fold increase over that in the VR1055 supernatant). In a second experiment, performed with different macrophage supernatants, production of 5.4- and 7.2-fold more IFN-{gamma} was induced by supernatants of macrophages stimulated with modified VR1055-CS pDNA than with supernatants of macrophages stimulated with VR1055-CSS or VR1055 pDNA, respectively (P =0.013). In this assay, unmodified and VR1055-CSS pDNA stimulated comparable levels of IFN-{gamma}-inducing activity (inducing 81 and 61 U/mL of IFN-{gamma}, respectively, compared with 42 U/mL of IFN-{gamma} induced by control macrophage supernatants). Thus, although the relative induction of IL-12-like bioactivity by unmodified or CSS-modified pDNA differed in the two experiments, in each experiment the CpG-modified pDNA induced significantly more IL-12 bioactivity than unmodified or non-CpG-modified pDNA.



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  		Figure 5. Modified VR1055-CS pDNA induces IL-12, TNF-{alpha}, and NO production. Macrophages were cultured for 24 h (A, B) or 48 h (C) with medium alone or with 25 µg/mL of pDNA or bovine DNA with IFN-{gamma} (50 U/mL) and polymyxin B sulfate (10 µg/mL). (A) Macrophage supernatants were cocultured with phytohemagglutinin-stimulated PBMCs for 48 h, and supernatants were assayed by ELISA for IFN-{gamma} production. Results are means ± 1 SD of duplicate determinations. Significantly more IFN-{gamma} was induced by supernatants of macrophages stimulated with VR1055 pDNA than by supernatants of untreated macrophages (P =0.0002, indicated by #). Significantly more IFN-{gamma} was induced by supernatants of macrophages stimulated with modified VR1055-CS pDNA than with supernatants of macrophages stimulated with unmodified VR1055 pDNA or pDNA VR1055-CSS (P< 0.005, indicated by *). These data are representative of two independent experiments. (B) TNF-{alpha} levels were determined by ELISA by comparison to a standard curve generated with recombinant bovine TNF-{alpha} (20–10,000 pg/mL). TNF-{alpha} concentrations were determined from the means ± 1 SD of values from duplicatereactions, and results presented are from one representative experiment of six. Significantly more TNF-{alpha} was produced by VR1055 pDNA-stimulated macrophages than by untreated macrophages (P <0.05, indicated by *). Significantly more TNF-{alpha} was produced by VR1055-CS pDNA-stimulated macrophages than by unmodified VR1055 pDNA-stimulated macrophages (P =0.01, indicated by #). (C) NO2- accumulation in macrophage supernatants was determined using the Griess assay. Results are the means ± 1 SD of values for triplicate cultures. Significantly more NO2- was produced by macrophages stimulated with VR1055-CS pDNA than by cells stimulated with any of the negative-control pDNAs (P <0.05, indicated by *). These data are representative of four independent experiments.

 
We next analyzed TNF-{alpha} production by ELISA. Unmodified VR1055 pDNA-stimulated macrophages produced significantly more (P =0.014) TNF-{alpha} than untreated macrophages (Fig. 5B) . Modified VR1055-CS pDNA treatment significantly augmented (P <0.01) TNF-{alpha} production over that induced by unmodified pDNA in six experiments using macrophages from different cattle and two different preparations of pDNA, whereas VR1055-CSS pDNA, containing the scrambled, non-CpG sequence of CS ODN, had no effect on TNF-{alpha} production. In five experiments, modified VR1055-CS pDNA induced four- to sixfold increases in TNF-{alpha} production over that induced by unmodified pDNA. A sixth experiment demonstrated a 15-fold enhancement. TNF-{alpha} was not detected in supernatants from macrophages exposed to bovine DNA.

Macrophage supernatants were also analyzed for the presence of NO2- as a measure of NO production. Macrophages treated with unmodified VR1055 pDNA reproducibly made more NO2- than did untreated macrophages. Consistent with the steady-state levels of iNOS mRNA, macrophages treated with modified VR1055-CS pDNA produced significantly (P <0.05) more NO2- than did those treated with unmodified VR1055 or VR1055-CSS pDNA or untreated macrophages (Fig. 5C) . These data are representative of four experiments performed with macrophages from different cattle and two different pDNA preparations. Approximately twofold increases in NO production by VR1055-CS plasmid DNA versus unmodified pDNA or VR1055-CSS pDNA were consistently observed.

The additional CpG motifs present in VR1055-CS pDNA fail to potentiate B-cell proliferation induced by unmodified VR1055 pDNA
Our results with ODN containing one or two AACGTT motifs demonstrated effective stimulation of B-cell proliferation [25 ] (Fig. 2A) . Furthermore, the addition of CpG motifs to pDNA has been shown to enhance antibody production in mice [19 , 20 ]. Therefore, the mitogenic activity of unmodified and modified pDNAs for bovine B cells was determined. B-cell proliferation induced by unmodified VR1055 pDNA was significantly increased over that of unstimulated B cells (Fig. 6 ). However, incorporation of two AACGTT motifs into pDNA did not further increase its ability to stimulate B-cell proliferation.



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  		Figure 6. Stimulation of B-cell proliferation by pDNA. B cells were cultured for 72 h with 3.125–25.0 µg/mL of pDNA in the presence of 10 µg/mL of polymyxin B sulfate. Results are means ± 1 SD of values for triplicate cultures. Unmodified VR1055 pDNA is indicated by the open squares, VR1055-CS pDNA is indicated by the closed squares, and medium is indicated by the dashed line. The asterisk indicates that proliferation was significantly increased over that induced by medium alone (P <0.05). These data are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies in our laboratory demonstrated a CpG-dependent activation of bovine B-lymphocyte proliferation and IgG2 production by B. bovis and E. coli DNAs and oligonucleotides containing the AACGTT motif [25 ]. More recent experiments have demonstrated activation of bovine macrophages by E. coli and protozoan parasite DNAs to express IL-12, NO, and TNF-{alpha} [26 ]. These humoral and cellular responses, which are characteristic of a type 1 response, are hypothesized to be critical for protection against the intracellular erythrocyte pathogens A. marginale and B. bovis [35 , 36 , 42 ]. One focus of our research is to identify ways to promote enhanced type 1 immunity against these pathogens during immunization [43 ]. Because of the adjuvant properties inherent in DNA vaccines, there is interest in optimizing such vaccines to target the desired immune response [44 ]. pDNA was shown to effect a type 1 response when B lymphocytes, macrophages, and dendritic cells of mice were studied [16 , 17 ], indicating that nucleic acid vaccines supply adjuvant effects as well as antigen priming. Strong evidence for the adjuvant properties of pDNA was first demonstrated by Sato et al. [19 ]. Incorporation of one or two AACGTT motifs into the noncoding region of the pDNA vector encoding ß-galactosidase caused a dose-dependent enhancement of humoral and cellular immune responses to the protein. Subsequently, up-regulation of IL-12, IL-18, and type I IFNs in human PBMCs transfected with pDNA containing the additional AACGTT motifs was demonstrated by the same group [18 ]. Studies performed by Klinman et al. [20 ] confirmed this effect by demonstrating an enhanced adjuvanticity of pDNA when three AACGTT motifs were physically linked to the pDNA (1012/PyCSP, encoding the circumsporozoite protein of Plasmodium yoelii). CpG-modified pDNA enhanced parasite-specific IgG responses by 10-fold and IFN-{gamma} responses by 4-fold in vaccinated mice. We have extended these studies by showing that bovine monocyte-derived macrophages cultured with pDNA in the absence of lipophilic polyamines are activated to express IL-1ß, TNF-{alpha}, IL-12, and NO. Importantly, the addition of two AACGTT motifs to the pDNA enhanced production of IL-12, TNF-{alpha}, and NO over that induced by unmodified pDNA, with TNF-{alpha} and NO determined to be present in statistically significantly higher levels in repeated experiments with two different pDNA preparations. The levels of enhancement by AACGTT-modified pDNA were approximately threefold for NO, two- to fourfold for IL-12 p40/p70, two- to sevenfold for IL-12-like activity, and three-to sixfold for TNF-{alpha}, values similar to those reported by others using AACGTT ISS-modified pDNA [18 19 20 ]. Some differences in the amount of cytokine induced by the different pDNAs were observed between experiments. We attribute this variation, which is typical of primary bovine macrophage assays [26 , 37 , 38 ], to different states of activation of the macrophage cultures. However, CpG-modified pDNA consistently stimulated significantly higher levels of cytokines and NO than did control pDNA.

We observed that in most assays, unmodified and VR1055-CS pDNAs were less stimulatory than E. coli DNA (Fig. 4 and data not shown). The reasons for these differences in immunostimulatory activity of pDNAs and E. coli DNA are not known. The superior stimulatory activity of E. coli DNA compared with other bacterial and protozoan DNAs has been observed by others [45 , 46 ] and by ourselves [26; Y. Zhang, W. C. Brown, unpublished observations]. A higher GC content and/or frequency of CG dinucleotides in the genome may partly explain the comparatively higher activity of E. coli DNA [26 , 47 ]. Whereas E. coli contains approximately the expected proportion (6.25%) of CG dinucleotides in its genome, less than the expected frequency of CG dinucleotides is present in protozoan-parasite DNA [25 , 26 , 47 ] and in VR1055 or VR1055-CS pDNAs (5.2%). The level of cytokine induction by E. coli DNA versus that of protozoan-parasite DNA strongly correlated with the CG dinucleotide frequency in the genome [26 ], supporting the possibility that pDNA is less active because it contains fewer CG dinucleotides than E. coli DNA. The stronger stimulation by E. coli DNA and VR1055-CS pDNA is not due to contaminating endotoxin. Although LPS and E. coli DNA may act synergistically, these agents stimulated human monocytes to secrete IL-6 and TNF with different kinetics, demonstrating a different mode of activation [23 ]. Importantly, in our experiments, pDNA preparations did not contain detectable endotoxin, and all studies were conducted in the presence of saturating amounts of polymyxin B sulfate that neutralized 1,000 pg/mL of LPS, which was 100-fold more than the level detected in E. coli DNA preparations (Fig. 1) .

We occasionally observed that the VR1055-CSS pDNA modified to contain the non-CpG insert was less stimulatory than unmodified pDNA (Fig. 5A and 5B) . Since the molecular mechanisms of sequence-dependent CpG DNA activation have not been completely elucidated, and the spacing of CpG motifs and presence of certain flanking residues in DNA may positively or negatively influence activity [3 , 4 ], the possibility that the presence of the additional GC sequence in VR1055-CSS pDNA results in an inhibitory effect cannot be excluded. In support of this possibility, a nonstimulatory ODN was shown to inhibit pDNA-induced proliferation of murine spleen cells [48 ].

There are three lines of evidence for enhancement of IL-12 production by modified pDNA. First, RT-PCR analysis demonstrated higher-level IL-12 p35 and IL-12 p40 mRNA accumulation in macrophages incubated with VR1055-CS than in those incubated with unmodified VR1055. Second, binding of an IL-12 p40-specific mAb indicated that VR1055-CS induced higher levels of IL-12 p40/p70 secretion than VR1055. Third, an IL-12 bioassay demonstrated the induction of increased IFN-{gamma} production by PBMCs cultured with supernatants from VR1055-CS-treated macrophages compared with that of PBMCs cultured with supernatants from unmodified-pDNA-treated macrophages. Collectively, these data indicate that VR1055-CS stimulates production of a biologically active IL-12 heterodimer. This could not be definitively demonstrated because recombinant bovine IL-12 is not available to determine the potential neutralizing capability of the IL-12 p40-specific mAb used for detection. Furthermore, we cannot eliminate the possibility of a contribution by IL-18, which is also produced by macrophages and acts independently of or synergistically with IL-12 to stimulate IFN-{gamma} production by bovine PBMCs and T cells [49 ]. Nevertheless, IL-18 is also a type I cytokine, so its induction would be consistent with the goal of achieving IFN-{gamma}-inducing adjuvant activity. Induction of IL-18 was not determined because of the lack of availability of biological or antibody assays to measure bovine IL-18. Furthermore, because IL-18 is posttranslationally modified by caspase-1 to its biologically active form, mRNA levels of this cytokine do not predict biological activity. Transcripts of IL-18 were expressed constitutively, and this expression was not up-regulated in activated bovine macrophages (49).

Relative to that induced with VR1055 pDNA, production of TNF-{alpha} and NO was reproducibly elevated after stimulation with VR1055-CS. NO is frequently produced in association with inflammatory cytokines such as TNF-{alpha}, and TNF-{alpha} induces NO production by murine and bovine macrophages [50 , 51 ]. Because TNF-{alpha} is involved in immune-cell recruitment and production of other inflammatory mediators, enhanced TNF-{alpha} production may be a desirable adjuvant property. However, the potential pathological consequences of excessive TNF-{alpha} production in response to DNA vaccines warrant consideration [45 ].

The ability of modified pDNA to induce IL-12 production has important implications with regard to the use of similarly modified pDNA vaccine vectors to immunize cattle against intracellular pathogens of cattle, such as A. marginale and B. bovis, because this cytokine is important for initiating type 1 immune responses, which are important for protection against such pathogens. IFN-{gamma}, produced by Th1 cells, primes macrophages for enhanced NO production, and both IFN-{gamma} and NO are critical components of protective immunity against Ehrlichia phagocytophila [52 , 53 ], the agent of human granulocytic ehrlichiosis, which is closely related to A. marginale. In addition, macrophage-derived NO partially inhibits replication of B. bovis [38 ].

The B-lymphocyte property of mitogenesis is one of the defining characteristics of CpG motifs [6 ]. In preliminary experiments, we found that, whereas linearized pDNA was less effective at activating macrophages, linearization of pDNA was required to induce B-lymphocyte proliferation (data not shown). Linearized, unmodified VR1055 pDNA stimulated significant B-lymphocyte proliferation that was not further enhanced by addition of AACGTT motifs. However, an ODN consisting of the sequence inserted into the VR1055-CS pDNA was stimulatory for B cells. Thus, one likely explanation for this result is that the CpG motifs inherent in the pDNA are adequate to stimulate B-cell proliferation [44 ]. In support of this possibility, the VR1055 pDNA contains one copy of the GACGTT motif, which was shown to stimulate bovine B cells when present as a single copy in an ODN [25 ]. Studies in our laboratory have also determined that the GTCGTT motif, which is present in triplicate in ODN 2059, is also stimulatory for bovine B cells, indicating that additional, nonclassical CpG motifs are active on B cells of cattle. Furthermore, ODN 2059 stimulates higher levels of B-cell proliferation than ODNs containing one or two copies of the AACGTT motif (Y. Zhang, W. C. Brown, unpublished observations), suggesting that the AACGTT motif may not be optimal for activating bovine B-cell responses.

In conclusion, the research presented here was undertaken to improve the adjuvant effect of a pDNA vector used to immunize cattle against A. marginale [33 ]. DNA vaccines have been employed with some success to protect cattle against viral pathogens [27 28 29 30 31 32 ]. However, there is clearly a need to better delineate the nature of the protective immune response and to design vaccines that amplify the desired elements of protection. Our data provide a basis for optimizing nucleic-acid-based immunization regimens to promote type 1 immune responses toward viral and intracellular protozoan and ehrlichial pathogens in cattle.


   ACKNOWLEDGEMENTS
 
This research was supported by USDA-NRICGP grants 98-35204-6462, 98-35204-6737, and 99-35204-8368.

We thank Monica Florin-Christensen and Nissa Gese for technical help and Dale Godson for providing reagents for the TNF-{alpha} ELISA.

Received September 30, 2000; revised November 11, 2000; accepted February 9, 2001.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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