DNA vaccination represents a novel means of expressing Ag in vivo for the generation of both humoral and cellular immune responses. The current study uses this technology to elicit protective immunity against experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease of the central nervous system that serves as an experimental model for multiple sclerosis. RT-PCR verified by Southern blotting and sequencing of PCR products of four different C-C chemokines, macrophage-inflammatory protein-1α (MIP-1α), monocyte-chemotactic protein-1 (MCP-1), MIP-1β, and RANTES, were performed on brain samples from EAE rats to evaluate mRNA transcription at different stages of disease. Each PCR product was then used as a construct for naked DNA vaccination. The subsequent in vivo immune response to MIP-1α or MCP-1 DNA vaccines prevented EAE, even if disease was induced 2 mo after administration of naked DNA vaccines. In contrast, administration of the MIP-1β naked DNA significantly aggravated the disease. Generation of in vivo immune response to RANTES naked DNA had no notable effect on EAE. MIP-1α, MCP-1, and MIP-1β mRNA transcription in EAE brains peaked at the onset of disease and declined during its remission, whereas RANTES transcription increased in EAE brains only following recovery. Immunization of CFA without the encephalitogenic epitope did not elicit the anti-C-C chemokine regulatory response in DNA-vaccinated rats. Thus, modulation of EAE with C-C chemokine DNA vaccines is dependent on targeting chemokines that are highly transcribed at the site of inflammation at the onset of disease.

Experimental autoimmune encephalomyelitis (EAE)3 is an autoimmune disease of the central nervous system (CNS) that serves as a model for the human disease multiple sclerosis, since in both diseases circulating leukocytes penetrate the blood brain barrier and damage myelin, resulting in impaired nerve conduction and paralysis (1, 2). We have previously used molecular biologic techniques to follow leukocyte trafficking to the site of inflammation at the CNS of EAE rats, and suggested a model that characterizes this process as a sequential multistep event (3). At first, a very limited repertoire of T cells, which we named the primary influx, interacts with their target Ag at the site of inflammation, leading to the activation of the blood brain barrier to express various adhesion molecules and thus to increase its permeability to circulating leukocytes (3, 4). Enhanced permeability of this barrier allows a nonselective influx of leukocytes, which we named the secondary influx. This influx correlates with disease onset (3, 5). Subsequently, Ag-specific autoimmune T cells either become anergic or undergo programmed cell death (apoptosis), leading to a remission in disease severity (6). Inhibition of the secondary influx, by either soluble peptide therapy or antiadhesion molecule blockade, effectively prevented, or even reversed, an ongoing disease even though the primary influx remained apparent at the site of inflammation (3, 4, 5, 7). Taken together, these results not only suggest novel therapeutic strategies, but also emphasize the important role of the nonselective leukocyte influx to a site of inflammation.

Chemokines are chemoattractants that mediate leukocyte attraction and recruitment at the site of inflammation. As so, they are likely to be key mediators in the recruitment of the secondary influx of leukocytes at an inflamed target organ. This has motivated us to use the novel technology of naked DNA vaccination (8, 9, 10, 11, 12, 13, 14, 15, 16, 17) and explore the therapeutic potential of anti-chemokine immunotherapy in EAE. Based on the positions of the first two cysteines, the chemokines can be divided into four highly conserved but distinct supergene families C-C, C-X-C, C, and the newly discovered C-X3-C (18, 19). The C-C family is involved primarily in the activation of endothelium and in the chemoattraction of T cells and monocytes to the site of inflammation (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). The protective competence of anti-C-C chemokine-based immunotherapy has been demonstrated by Karpus et al., who blocked EAE in mice by immunizing them with rabbit anti-mouse polyclonal Abs against macrophage-inflammatory protein-1α (MIP-1α) (33), and very recently by Gong et al., who used an antagonist of monocyte-chemoattractant protein-1 (MCP-1) to inhibit arthritis in the MRL-lpr mouse model (34). In another study, Berman et al. used in situ hybridization to demonstrate the dominant expression of MCP-1 in rat EAE brain (35). In the current study, we have cloned each of the major C-C chemokines, MCP-1, MIP-1α, MIP-1β, and RANTES, from EAE brains into an eukaryotic expression vector and determined their capacity to block EAE.

Female Lewis rats, approximately 6 wk old, were purchased from Harlan (Jerusalem, Israel) and maintained under specific pathogen-free conditions in our animal facility.

Myelin basic protein (MBP) p68–86, Y G S L P Q K S Q R S Q D E N P V, was synthesized on a MilliGen 9050 peptide synthesizer by standard 9-fluorenylmethoxycarbonyl chemistry. Peptides were purified by HPLC. Structure was confirmed by amino acid analysis and mass spectroscopy. Only peptides that were >95% pure were used in our study.

Rats were immunized s.c. in the hind footpads with 0.1 ml of MBP epitope 68–86 (p68–86) dissolved in PBS (1.5 mg/ml) and emulsified with an equal volume of CFA (in CFA supplemented with 4 mg/ml heat-killed Mycobacterium tuberculosis H37Ra in oil) (Difco, Detroit, MI). Rats were then monitored daily for clinical signs by an observer blind to the treatment protocol. EAE was scored as follows: 0, clinically normal; 1, flaccid tail; 2, hind limb paralysis; and 3, front and hind limb paralysis.

At 9 days after induction of active EAE, draining lymph node cells were cultured (12 × 106/ml) for 3 days in stimulation medium that includes DMEM (Life Technologies, Gaithersburg, MD) supplemented with 2-ME (5 × 10−5 M), l-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (100 μ/ml), streptomycin (100 μg/ml), 1% syngeneic serum, and 20 to 30 μg/ml of the immunizing epitope; washed; and resuspended in resting medium, which was identical to the stimulation medium without syngeneic serum and with the addition of 10% FCS (Life Technologies) and 12.5% supernatant of Con A-stimulated splenocytes as a source of T cell growth factors. Con A supernatant was prepared as described elsewhere (36). After 5 to 7 days in resting medium, the cells (5 × 105/ml) were activated for 3 days in the presence of irradiated (2500 rad) syngeneic thymocytes (12 × 106/ml) and 10 to 20 μg/ml of p68–86. The activated T cells were then either used for induction of transferred EAE or resuspended in resting medium for additional growing cycles.

Transferred EAE was induced by immunizing Lewis rats (i.p.) with 107 in vitro-activated (day 3) L68–86 cells.

RT-PCR analysis, verified by Southern blotting, was utilized on brain samples, according to the protocol we described elsewhere, with some modifications (3). Rats were euthanized by CO2 narcosis. Brain samples containing mainly the midbrain and brain stem were obtained after perfusion of the rat with 160 to 180 ml of ice-cold PBS injected into the left ventricle following an incision in the right atrium. Each sample was homogenized. Total RNA was extracted using the Tri-Zol procedure (Life Technologies), according to the manufacturer’s protocol. mRNA was then isolated using a mRNA isolation (kit 1741985; Boheringer Mannheim, Mannheim, Germany), and reverse transcribed into first strand cDNA exactly as we have described in detail elsewhere (3). First strand cDNA was then subjected to 35 cycles of PCR amplification using specific oligonucleotide primers that we designed based on the published sequence of each chemokine (NCBI accession numbers: rat MIP-1α, U06435; rat MIP-1β, U06434; rat RANTES, U06436; and rat MCP-1, M57441), as follows: rat MIP-1α sense, 5′-ATGAAGGTCTCCACCACTGCCCTTGC-3′; rat MIP-1α antisense, 5′-TCAGGCATTCAGTTCCAGCTCAGTG-3′; rat MIP-1β sense, 5′-ATGAAGCTCTGCGTGTCTGCCTTC-3′; rat MIP-1β antisense, 5′-TCAGTTCAACTCCAAGTCATTCAC-3′; rat RANTES sense, 5′-ATGAAGATCTCTGCAGCTGCATCC-3′; rat RANTES antisense, 5′-CTAGCTCATCTCCAAATAGTTG-3′; rat MCP-1 sense, 5′-ATGCAGGTCTCTGTCACGCTTCTGGGC-3′; and rat MCP-1 antisense, 5′-CTAGTTCTCTGTCATACTGGTCAC.

All RNA samples were calibrated to β-actin: rat β-actin sense, 5′-CATCGTGGGCCGCTCTAGGCA-3′; rat β-actin antisense, 5′-CCGGCCAGCCAAGTCCAGACG-3′.

The cycle profile was: denaturation at 95°C for 60 s, annealing at 55°C for 60 s, and elongation at 72°C for 60 s. Amplified products were subjected to electrophoresis, transferred to nylon membranes (MagnaGraph nylon transfer membrane, msi; Westborough, MA), fixed with UV light (120 mjoules), and hybridized with 106 cpm/ml of α-32P-labeled DNA fragments encoding the full-length PCR product of each C-C chemokine and of β-actin (random priming; Amersham, Arlington Heights, IL). PCR products were used as probes only after each PCR product was cloned and its sequence was verified as described below.

Each of the amplified PCR products described above was cloned into a pUC57/T vector (T-cloning kit K1212; MBI Fermentas, Vilnius, Lithuania) and transformed to Escherichia coli, according to the manufacturer’s protocol. Each clone was then sequenced (Sequenase version 2; United States Biochemical, Cleveland, OH) according to the manufacturer’s protocol. PCR products were selected to be used as constructs for naked DNA vaccination only after cloning and sequence verification.

DNA vaccination was performed according to Waisman et al., with some modifications (17). Sequenced PCR products of rat MIP-1α, MCP-1, MIP-1β, and RANTES were transferred into a pcDNA3 vector (Invitrogen, San Diego, CA). Large scale preparation of plasmid DNA was conducted using Mega prep (Qiagen, Chatsworth, CA). Cardiotoxin (Sigma, St. Louis, MO) was injected into the tibialis anterior muscle of 6- to 8-wk-old female Lewis rats (10 μM per leg). At 1 wk following injection, rats were injected with 100 μg DNA in PBS. At 4 to 5 days after the first immunization, one rat from each group was sacrificed, and transcription of the relevant chemokine was verified using RT-PCR on tibialis anterior muscle samples. Thereafter, naked DNA vaccines were given three to five times, with intervals of 6 to 7 days between each injection.

PCR products were recloned into a PQE expression vector (PQE-30, PQE-31, or PQE-32, according to the correct open reading frame), expressed in E. coli (Qiagen), and then purified by an NI-NTA-superflow affinity purification of 6xHis proteins (Qiagen). Each recombinant protein sequence has been verified (N terminus) by our sequencing services unit.

Abs from rat sera were purified using a High-Trap protein G column (Pharmacia, Piscataway, NJ), according to the manufacturer’s protocol. Then Ab titer to various chemokines was determined by an ELISA assay, as described below.

In vitro chemotaxis assay was conducted as described previously by one of us (37), with minor modifications (according to 38 . Peritoneal macrophages were isolated as described previously (38) and suspended in DMEM enriched with 1% BSA. Cell migration was evaluated in standard Boyden chambers (Neuroprobe, Cabin John, MD). Macrophages (1.2 × 106 cells) were added to the upper well. Chemotactic factors FMLP (Sigma; 10−7 M), rat rMIP-1α (Chemicon International, Temecula, CA; 200 ng/ml), or rat rMCP-1 (Chemicon; 100 ng/ml) were added to the lower wells with or without preincubation with the required Abs (10 μg/well) at 37°C for 30 min. Migration was allowed to proceed for 90 min at 37°C. The cellulose nitrate filters (5 μm pore size) were then fixed and stained as described previously (37). A total of 5 ×400 fields were selected randomly on each filter, and the number of migrating cells was counted.

A direct ELISA assay has been utilized to determine the anti-C-C chemokine Ab titer in DNA-vaccinated rats. Each recombinant chemokine that we produced, as well as commercial recombinant rat RANTES, rat MIP-1α, rat MCP-1, and human MIP-1β (Chemicon) were coated onto 96-well ELISA plates (Nunc, Roskilde, Denmark), at concentrations of 50 ng/well. Rat antisera, in serial dilutions from 28 to 230 were added to ELISA plates coated with each recombinant chemokine. Goat anti-rat IgG alkaline phosphatase-conjugated Abs (Sigma) were used as a labeled Ab. p-Nitrophenyl phosphate (p-NPP; Sigma) was used as a soluble alkaline phosphatase substrate. The assay conditions and data calculation of each test were performed according to Reference 14. Results are shown as log2 Ab titer ± SE. Commercial mAbs to each chemokine (Chemicon) were used as a positive control for detected sera in each experiment.

Histologic examination of hematoxylin and eosin-stained sections of Formalin-fixed, paraffin-embedded sections of brain and the lower thoracic and lumbar regions of the spinal cord was performed. Each section was evaluated without knowledge of the treatment status of the animal. The following scale was used: 0, no mononuclear cell infiltration; 1, 1 to 5 perivascular lesions per section with minimal parenchymal infiltration; 2, 5 to 10 perivascular lesions per section with parenchymal infiltration; and 3, >10 perivascular lesions per section with extensive parenchymal infiltration. The mean histologic score ± SE was calculated for each treatment group. A representative photomicrograph is shown in Figure 3.

FIGURE 3.

MIP-1α and MCP-1 naked DNA vaccines decrease CNS mononuclear cell infiltration (see Table I).

FIGURE 3.

MIP-1α and MCP-1 naked DNA vaccines decrease CNS mononuclear cell infiltration (see Table I).

Close modal

Significance of differences was examined using the Student t test. A value of p < 0.05 was considered significant. One-way multiple range ANOVA test with significance level of p < 0.05 was performed for multiple compression of Ab titer to various C-C chemokines in naked DNA-vaccinated rats (Figs. 4 and 6).

FIGURE 4.

Synchronic protective immunity to EAE following C-C chemokine naked DNA vaccination. Twelve days after active induction of disease (with p68–86/CFA), sera of rats from experiment 2 (Fig. 2 B), as well as sera from rats that received the same subsequent set of naked DNA vaccinations, but were finally challenged with the emulsion of PBS and CFA without p68–86, or from rats that received the same subsequent set of naked DNA vaccinations, but were never challenged with p68–86/CFA or CFA, were tested for Ab titer against each of the four C-C chemokines: MCP-1 (A), MIP-1α (B), MIP-1β (C), and RANTES (D). The assay conditions and data calculation of each test were done (according to Ref. 14). Results are shown as mean log2 of four different samples ± SE.

FIGURE 4.

Synchronic protective immunity to EAE following C-C chemokine naked DNA vaccination. Twelve days after active induction of disease (with p68–86/CFA), sera of rats from experiment 2 (Fig. 2 B), as well as sera from rats that received the same subsequent set of naked DNA vaccinations, but were finally challenged with the emulsion of PBS and CFA without p68–86, or from rats that received the same subsequent set of naked DNA vaccinations, but were never challenged with p68–86/CFA or CFA, were tested for Ab titer against each of the four C-C chemokines: MCP-1 (A), MIP-1α (B), MIP-1β (C), and RANTES (D). The assay conditions and data calculation of each test were done (according to Ref. 14). Results are shown as mean log2 of four different samples ± SE.

Close modal
FIGURE 6.

Vaccination with MCP-1 DNA elicited a significant cross-reactive immune response to MIP-1α. Twelve days after active induction of disease, sera of rats from experiment 2 that were immunized with various C-C chemokine DNA vaccines and then challenged with p68–86/CFA (Figs. 2 B and 4) were tested for the development of a cross-reactive Ab titer between each of the four C-C chemokines: MCP-1, MIP-1α, MIP-1β, and RANTES. Results are shown as mean log2 of four different samples ± SE.

FIGURE 6.

Vaccination with MCP-1 DNA elicited a significant cross-reactive immune response to MIP-1α. Twelve days after active induction of disease, sera of rats from experiment 2 that were immunized with various C-C chemokine DNA vaccines and then challenged with p68–86/CFA (Figs. 2 B and 4) were tested for the development of a cross-reactive Ab titer between each of the four C-C chemokines: MCP-1, MIP-1α, MIP-1β, and RANTES. Results are shown as mean log2 of four different samples ± SE.

Close modal

Rats injected with L68–86 developed transferred EAE that persisted for 5 to 6 days (Fig. 1,A). Before adoptive transfer of disease (day 0), and at various time points, before the onset of disease (day 3), at the day of onset (day 5), the peak (day 7), following recovery (day 10), and 10 days after recovery (day 20), midbrain-brain stem samples were obtained from six different rats at each time point. From each sample, mRNA was isolated and subjected to RT-PCR analysis using specific oligonucleotide primers that we constructed to each chemokine. Each amplification was calibrated to β-actin and verified by Southern blotting analysis. This enabled semiquantitative analysis of the dynamics of mRNA transcription of each of the above C-C chemokines at the site of inflammation. Figure 1,B shows representative results from each time point of the experiment. An increased transcription of MIP-1α, MCP-1, and MIP-1β mRNA in EAE brains was observed at the onset of disease (day 5). The augmented transcription of MIP-1α and MCP-1 regressed to background within 2 days, even though disease continued to progress to its maximal clinical score on day 7 (Fig. 1, A and B). The increased transcription of MIP-1β, however, declined to its background in correlation with recovery (Fig. 1, A and B). Unexpectedly, RANTES transcription increased in EAE brains only after recovery. The biologic significance of this observation remains to be elucidated.

FIGURE 1.

Dynamics of mRNA transcription of various C-C chemokines in the inflamed brain. A and B, Rats were injected with 107 L68–86 cells to develop transferred EAE (A). Before adoptive transfer of disease (day 0), and at various time points, before the onset of disease (day 3), at the day of onset (day 5), the peak (day 7), following recovery (day 10), and 10 days after recovery (day 20), midbrain and brain stem samples from six different rats at each time point were examined. mRNA was isolated from each sample and subjected to RT-PCR analysis using specific oligonucleotide primers constructed to RANTES, MIP-1α, MIP-1β, and MCP-1. Each amplification was calibrated to β-actin and verified by Southern bloting analysis. B, A representative Southern blot analysis of each time point. C and D, Rats were immunized with p68–86/CFA and developed active EAE (C). Before the induction of disease (day 0), and at various time points, before the onset of disease (day 8), at the peak (day 13), and 5 days after recovery (day 21), midbrain and brain stem samples from six different rats at each time point were obtained and subjected to RT-PCR, as described above. D, A representative Southern blot analysis from each time point.

FIGURE 1.

Dynamics of mRNA transcription of various C-C chemokines in the inflamed brain. A and B, Rats were injected with 107 L68–86 cells to develop transferred EAE (A). Before adoptive transfer of disease (day 0), and at various time points, before the onset of disease (day 3), at the day of onset (day 5), the peak (day 7), following recovery (day 10), and 10 days after recovery (day 20), midbrain and brain stem samples from six different rats at each time point were examined. mRNA was isolated from each sample and subjected to RT-PCR analysis using specific oligonucleotide primers constructed to RANTES, MIP-1α, MIP-1β, and MCP-1. Each amplification was calibrated to β-actin and verified by Southern bloting analysis. B, A representative Southern blot analysis of each time point. C and D, Rats were immunized with p68–86/CFA and developed active EAE (C). Before the induction of disease (day 0), and at various time points, before the onset of disease (day 8), at the peak (day 13), and 5 days after recovery (day 21), midbrain and brain stem samples from six different rats at each time point were obtained and subjected to RT-PCR, as described above. D, A representative Southern blot analysis from each time point.

Close modal

Rats with developing active disease manifested similar mRNA transcription characteristics as those with developing transferred disease, that is, an elevated expression of MCP-1, MIP-1α, and MIP-1β at the onset of disease, which declines during recovery, and an augmented transcription of RANTES following recovery (Fig. 1, C and D).

Cloned PCR products of each C-C chemokine, obtained as described above, were ligated into a pcDNA3 eukaryotic expression vector and used as constructs for naked DNA vaccination (Fig. 2). In the first experiment (Fig. 2,A), rats were subjected to three weekly injections of each construct. Control rats were either injected with the pcDNA3 vector alone, or with PBS. Two weeks after the last immunization, all rats were immunized with p68–86/CFA to induce active EAE. All control (PBS-immunized) and pcDNA3-vaccinated rats developed active disease that persisted for 5 to 6 days (Fig. 2,A, 6/6 in each group with a maximum clinical score 2.33 ± 0.1 in control and 2 ± 0.26 in pcDNA3-immunized rats). In contrast, rats injected with either MIP-1α or MCP-1 DNA naked DNA vaccines were resistant to EAE (incidence of 0/6 for MIP-1α and 1/6 for MCP-1-vaccinated rats with a maximum clinical score of 0 and 0.33 ± 0.34, respectively; p < 0.001 for each treatment compared with either control or pcDNA3 treatments). Thus, the subsequent in vivo immune response to MIP-1α or MCP-1 DNA vaccines prevented EAE. In contrast, administration of the MIP-1β naked DNA significantly aggravated active EAE (Fig. 2 A, 6/6 in each group, maximum clinical score 3.2 ± 0.17 compared with 2.33 ± 0.1 in control and 2 ± 0.26 in pcDNA3-immunized rats; p < 0.033 and 0.028, respectively).

FIGURE 2.

Prevention of EAE using C-C chemokine naked DNA vaccines. Rats were immunized weekly (three repeated immunizations, A; five repeated immunizations, B) with the cloned PCR products of various C-C chemokines ligated into a pcDNA3 eukaryotic expression vector, or with the pcDNA3 vector alone, or with PBS. Two weeks after the last immunization, all rats were immunized with p68–86/CFA to induce active EAE. One month after the last immunization, all rats were immunized with p68–86/CFA to induce active EAE and monitored for clinical signs daily by an observer blind to the treatment protocol. EAE was scored as follows: 0, clinically normal; 1, flaccid tail; 2, hind limb paralysis; 3, front and hind limb paralysis. Results are shown as mean clinical score of six rats in each group ± SE.

FIGURE 2.

Prevention of EAE using C-C chemokine naked DNA vaccines. Rats were immunized weekly (three repeated immunizations, A; five repeated immunizations, B) with the cloned PCR products of various C-C chemokines ligated into a pcDNA3 eukaryotic expression vector, or with the pcDNA3 vector alone, or with PBS. Two weeks after the last immunization, all rats were immunized with p68–86/CFA to induce active EAE. One month after the last immunization, all rats were immunized with p68–86/CFA to induce active EAE and monitored for clinical signs daily by an observer blind to the treatment protocol. EAE was scored as follows: 0, clinically normal; 1, flaccid tail; 2, hind limb paralysis; 3, front and hind limb paralysis. Results are shown as mean clinical score of six rats in each group ± SE.

Close modal

MIP-1α, MCP-1, and MIP-1β mRNA transcription in EAE brains peaked at the onset of disease and declined during its remission, whereas RANTES transcription increased in EAE brains only following recovery (Fig. 1). Thus, intervention in EAE development by C-C chemokine DNA vaccines is effective provided that the related chemokine is highly transcribed at the site of inflammation at the onset of disease.

In a subsequent experiment, each of the above constructs, as well as pcDNA3 alone, was administered five rather than three times (Fig. 2,B). As with the first experiment, MIP-1α and MCP-1 naked DNA vaccines effectively prevented the development of active EAE (incidence of 1/6 for each treatment with a maximum clinical score of 0.17 ± 0.17, compared with 6/6 in either control or pcDNA-treated rats; p < 0.001 for each comparison); MIP-1β vaccine significantly aggravated the disease (Fig. 2 B, 6/6 in each group, maximum clinical score 3 ± 0 compared with 2 ± 0 in control and 1.33 ± 0.21 in pcDNA3-immunized rats; p < 0.001 for each comparison); and RANTES naked DNA vaccination did not exhibit any notable effect on disease manifestation. Five consecutive immunizations of pcDNA3 did, however, notably affect disease severity (maximum score in control rats 2 ± 0 compared with 1.33 ± 0.21 in pcDNA-treated rats, p < 0.007). It is possible that numerous subsequent immunizations of a eukaryotic vector with a viral promoter may affect cytokine production by T cells, as has recently been suggested (39).

When active EAE attained its maximal severity (day 12, Fig. 2,B), spinal cord samples of representative animals from each group (Expt. 2) were evaluated histologically (Table I, Fig. 3). While control EAE rats and rats previously immunized with pcDNA3 all displayed perivascular lesions with parenchymal mononuclear cell infiltration (Fig. 3, B and C, and Table I, B and C; mean histologic score 2.2 ± 0.3 and 1.8 ± 0.2, respectively), rats previously immunized with MIP-1α or MCP-1 naked DNA vaccines were either free of mononuclear cell infiltration, or exhibited minimal parenchymal infiltration (Fig. 3, D and E, and Table I, D and E, compared with Fig. 3, B and C, and Table I, B and C; mean histologic score 0.2 ± 0.2 and 0.4 ± 0.24 compared with 1.8 ± 0.2 and 2.2 ± 0.3; p < 0.001). In contrast, rats that were immunized with MIP-1β naked DNA vaccines manifested an extensive parenchymal mononuclear cell infiltration (Fig. 3,F and Table I F, mean histologic score 3 ± 0). Thus, inhibition or exacerbation of disease by various naked DNA vaccines could each be demonstrated histologically.

Table I.

MIP1-α and MCP-1 naked DNA vaccines decrease CNS mononuclear cell infiltration

TreatmentaEAEMean Histological Scoreb
— − 0 ± 0 
— 2.2 ± 0.3 
pcDNA3 alone 1.8 ± 0.2 
pcDNA3/MCP-1 0.2 ± 0.2c 
pcDNA3/MIP-1α 0.4 ± 0.24c 
pcDNA3/MIP-1β 3 ± 0d 
pcDNA3/RANTES 1.8 ± 0.2 
TreatmentaEAEMean Histological Scoreb
— − 0 ± 0 
— 2.2 ± 0.3 
pcDNA3 alone 1.8 ± 0.2 
pcDNA3/MCP-1 0.2 ± 0.2c 
pcDNA3/MIP-1α 0.4 ± 0.24c 
pcDNA3/MIP-1β 3 ± 0d 
pcDNA3/RANTES 1.8 ± 0.2 
a

Rats were treated as described in the legend to Figure 2 B.

b

When active EAE attained its maximal clinical severity (day 12, Expt. 2, Fig. 2 B), samples from the lower thoracic and lumbar regions of the spinal cord were evaluated histologically. Histological scores were determined using a 0 to 3 scale as described in Materials and Methods. The mean clinical score ± SE was calculated from six sections per spinal cord of two representative rats from each group.

c

p < 0.001 for D and E compared with either B or C.

d

p < 0.001 for F compared with either B or C.

The development of anti-self-protective immunity in DNA-vaccinated rats was evaluated. When active EAE attained its maximal severity (day 12; Fig. 2,B), blood samples of all animals that were sacrificed for histologic evaluation (Expt. 2, Table I, Fig. 3) were analyzed for the production of Abs against gene products of each vaccinated DNA (Fig. 4), for the kinetics of Ab production along the course of active disease (Fig. 5), and for the possible development of cross-reactive immunity between various chemokines (Fig. 6).

FIGURE 5.

Kinetics of Ab production in sera of EAE rats following C-C chemokine naked DNA vaccination. Rats were immunized weekly (three repeated immunizations) with the cloned PCR products of various C-C chemokines ligated into a pcDNA3 eukaryotic expression vector, as described in Figure 2,A. Two weeks after the last immunization, all rats were immunized with p68–86/CFA to induce active EAE. At different time points (0, 3, 5, 7, 10, 12, 21, 30, and 40 days after EAE induction), generation of anti-self Ab titer was determined as described in Figure 4. Results are shown as mean log2 of four different samples ± SE.

FIGURE 5.

Kinetics of Ab production in sera of EAE rats following C-C chemokine naked DNA vaccination. Rats were immunized weekly (three repeated immunizations) with the cloned PCR products of various C-C chemokines ligated into a pcDNA3 eukaryotic expression vector, as described in Figure 2,A. Two weeks after the last immunization, all rats were immunized with p68–86/CFA to induce active EAE. At different time points (0, 3, 5, 7, 10, 12, 21, 30, and 40 days after EAE induction), generation of anti-self Ab titer was determined as described in Figure 4. Results are shown as mean log2 of four different samples ± SE.

Close modal

Rats, without DNA vaccination with developing EAE, display a notable anti-self Ab titer to various C-C chemokines (to MCP-1, Fig. 4,A, 12.25 ± 0.55 versus 7 ± 0.47; to MIP-1α, Fig. 4,B, 11 ± 0.47 versus 8.25 ± 0.55; to MIP-1β, Fig. 4,C, 11 ± 0.47 versus 7 ± 0.47; to RANTES, Fig. 4,D, 11.25 ± 0.55 versus 7 ± 0.47), which is, however, not sufficient to prevent the development of disease. Naked DNA vaccination, on the other hand, significantly augmented this Ab titer (Fig. 4, A–D, 14.5 ± 0.33, 14.75 ± 0.55, 16.25 ± 0.72, and 17 ± 0.66 in rats immunized with MCP-1, MIP-1α, MIP-1β, or RANTES constructs versus 9 ± 0.47, 8.5 ± 0.33, 8.75 ± 0.29, and 7 ± 0.47 in rats immunized with pcDNA alone, p < 0.05 for each reciprocal comparison, and versus 7 ± 0.47, 8.25 ± 0.55, 7 ± 0.47, and 7 ± 0.47 in naive rats, p < 0.05 for each reciprocal comparison; no significant difference was identified between rats immunized with pcDNA alone and naive controls). Nevertheless, the Ab titer in rats immunized with each C-C chemokine DNA except RANTES markedly increased following induction of active EAE, but not following an immunization with CFA without p68–86 (Fig. 4, A–D, 21.25 ± 0.98, 19.25 ± 0.73, 21.25 ± 0.99, and 20 ± 0.47 in rats immunized with MCP-1, MIP-1α, MIP-1β, or RANTES constructs, and then with p68–86/CFA versus 14.75 ± 0.55, 13.75 ± 0.72, 15.25 ± 0.55, and 17.5 ± 1.1 in rats immunized with MCP-1, MIP-1α, MIP-1β, or RANTES constructs, and then with CFA, p < 0.05, for each reciprocal comparison except for the last one, 0.05 < p < 0.1). Thus, naked DNA vaccines may serve as a powerful technique to generate protective immunity against autologous cytokines and provides a tool by which the immune system is encouraged to elicit anti-self-protective immunity to restrain its own harmful reactivity only when such a response is needed.

Sera from each of the above groups, immunized with various DNA vaccines and then with p68–86/CFA, were analyzed for a possible development of cross-reactive Ab titer (Fig. 6). Sera from MIP-1α, MIP-1β, and RANTES DNA-vaccinated rats manifested a highly specific titer against homologous Ag (p < 0.05 for the compression of each titer to any of the other three chemokines). MCP-1-vaccinated rats, however, exhibited a significant cross-reactive Ab titer against MIP-1α (Fig. 6 B; 21.25 ± 0.99 to self, 17 ± 0.66 to MIP-1α, and 11 ± 0.47 to either MIP-1β or RANTES; p < 0.005 for the comparison of anti-MIP-1α with anti-MIP-1β or RANTES, Ab titer, and for the comparison of anti-self with anti-MIP-1α Ab titer). Since both MCP-1 and MIP-1α naked DNA vaccines are protective, it is possible that the protective immunity generated by anti-MCP-1 DNA vaccination may be mediated at least in part by reaction with MIP-1α.

Since DNA vaccination elicits both cellular and humoral responses against products of a given construct, it is difficult to know which of these responses contributed more to the development of EAE resistance in MCP-1 and MIP-1α DNA-vaccinated rats. To evaluate the possible contribution of anti-self Abs to the development of EAE resistance, 12 days after active induction of EAE, when production of anti-self Abs in naked DNA-vaccinated rats attained at its maximal titer (Fig. 5), Abs were purified (IgG fraction, protein G purification) and evaluated for their competence to inhibit the migration of oil-induced peritoneal macrophages in a Boyden chemotaxis chamber assay, as previously described by one of us (37). MCP-1- and MIP-1α-specific Abs produced in MCP-1 naked DNA-vaccinated rats significantly blocked MCP-1- and MIP-1α-induced chemotaxis (Table II, 70 ± 7 and 88 ± 12 versus 185 ± 15, p < 0.001 for each comparison), whereas MIP-1α-specific Abs generated in MIP-1α naked DNA-vaccinated rats effectively blocked MIP-1α-induced chemotaxis (63 ± 4 versus 155 ± 15, p < 0.001), and to a much lesser extent MCP-1-induced chemotaxis (144 ± 11 versus 185 ± 15, p < 0.05, Table II). Thus, MCP-1 and MIP-1α chemokine-specific Abs generated in naked DNA-vaccinated rats are neutralizing Abs. These Abs were then evaluated for their competence to provide subsequent protection from severe EAE (Fig. 7). Four days before the onset of active EAE, rats were daily challenged (days 6–13) with 100 μg of each of the above neutralizing Abs, or with Abs from rats that were vaccinated with pcDNA3 alone. Repeated administration of Abs from MCP-1 and from MIP-1α DNA-vaccinated rats provided substantial protection from disease progression (mean maximal score of 0.66 ± 0.2 in rats treated with purified Abs from either MCP-1 or MIP-1α DNA-vaccinated donors versus 3.16 ± 0.2 and 3 ± 0 in rats treated with purified Abs from PBS- or pcDNA3-treated rats, p < 0.001 for each compression). In addition, elevated levels of MCP-1- and MIP-1α-specific Abs could be observed in spinal cord fluid (SCF) of EAE rats (day 12 of active EAE) that were previously subjected to MCP-1 or MIP-1α naked DNA vaccines (log2 Ab titer of 27 ± 3 and 18 ± 2 to MCP-1 and MIP-1α in SCF of rats administered with MCP-1 naked DNA vaccine, and of 25 ± 3 to MIP-1α in SCF of rats administered with MIP-1α naked DNA vaccine, compared with 12 ± 2 and 10 ± 1 in SCF of rats treated with pcDNA3 or PBS, p < 0.01 for each comparison). Rats administered with MIP-1α naked DNA vaccine did not generate a significant Ab titer to MCP-1 compared with rats administered with pcDNA3 or PBS. Thus, during the course of EAE, neutralizing Abs to MCP-1 and MIP-1α are generated in MCP-1 and MIP-1α DNA-vaccinated rats, and elevated levels of these Abs can be identified at the site of inflammation in the CNS, where they probably block disease progression.

Table II.

Abs from MIP-1α and MCP-1 naked DNA-vaccinated rats block MIP-1α- and MCP-1-induced chemotaxis in vitroa

ChemoattractantPurified Abs (IgG) from
Control EAE ratsMIP-1αb DNA-vaccinated EAE rats (cells/field ± SE)MCP-1b DNA-vaccinated EAE ratspcDNA3b DNA-vaccinated EAE rats
Medium 60 ± 6 66 ± 8 62 ± 4 57 ± 5 65 ± 6 
FMLP (10−7 M) 220 ± 14 213 ± 17 215 ± 17 211 ± 17 211 ± 19 
MIP-1α (200 ng/ml) 155 ± 15 143 ± 10 63 ± 4c 88 ± 12c 144 ± 11 
MCP-1 (100 ng/ml) 185 ± 15 179 ± 12 144 ± 11d 70 ± 7c 173 ± 10 
ChemoattractantPurified Abs (IgG) from
Control EAE ratsMIP-1αb DNA-vaccinated EAE rats (cells/field ± SE)MCP-1b DNA-vaccinated EAE ratspcDNA3b DNA-vaccinated EAE rats
Medium 60 ± 6 66 ± 8 62 ± 4 57 ± 5 65 ± 6 
FMLP (10−7 M) 220 ± 14 213 ± 17 215 ± 17 211 ± 17 211 ± 19 
MIP-1α (200 ng/ml) 155 ± 15 143 ± 10 63 ± 4c 88 ± 12c 144 ± 11 
MCP-1 (100 ng/ml) 185 ± 15 179 ± 12 144 ± 11d 70 ± 7c 173 ± 10 
a

At 12 days after active induction of EAE, when production of anti-self Abs in naked DNA-vaccinated rats attained at its maximal titer (Fig. 5), Abs were purified (IgG fraction, protein G purification) and evaluated for their competence in inhibiting the migration of oil-induced peritoneal macrophages in a Boyden chemotaxis chamber assay. FMLP (Sigma) was used as a positive control for chemoattraction. Results are shown as the mean of triplicates ± SE.

b

Donor rats were treated as described in the legend to Figure 2 A.

c

P < 0.001.

d

P < 0.05.

FIGURE 7.

Anti-chemokine Abs produced by DNA vaccination provide subsequent protection from severe EAE. Six groups of six rats each were immunized with p68–86/CFA to develop active EAE. Four days before the onset of disease, rats were daily challenged (i.v., days 6–13) with 100 μg of each of neutralizing Abs (IgG fraction, protein G purification) purified from sera of rats that were previously vaccinated with various naked DNA vaccines, and were then subjected to active induction of EAE, as described in Figure 2 A. Purified IgG fraction from rats that were or were not vaccinated with pcDNA3 and then subjected to active induction of disease, as well as sera from control rats that were or were not subjected to active induction of EAE were all used as controls. EAE was monitored daily by an observer blind to the treatment protocol. Results are shown as mean clinical score of six rats in each group ± SE.

FIGURE 7.

Anti-chemokine Abs produced by DNA vaccination provide subsequent protection from severe EAE. Six groups of six rats each were immunized with p68–86/CFA to develop active EAE. Four days before the onset of disease, rats were daily challenged (i.v., days 6–13) with 100 μg of each of neutralizing Abs (IgG fraction, protein G purification) purified from sera of rats that were previously vaccinated with various naked DNA vaccines, and were then subjected to active induction of EAE, as described in Figure 2 A. Purified IgG fraction from rats that were or were not vaccinated with pcDNA3 and then subjected to active induction of disease, as well as sera from control rats that were or were not subjected to active induction of EAE were all used as controls. EAE was monitored daily by an observer blind to the treatment protocol. Results are shown as mean clinical score of six rats in each group ± SE.

Close modal

To further evaluate a possible association between disease manifestation and anti-self Ab production in naked DNA-vaccinated rats, the kinetics of anti-self Ab was carefully evaluated. Rats have been subjected to MCP-1, MIP-1α, MIP-1β, or RANTES naked DNA vaccines and then immunized with p68–86/CFA, as described in the legend to Figure 2,A. At different time points (0, 3, 5, 7, 10, 12, 21, 30, and 40 days after EAE induction), generation of anti-self Ab was determined (Fig. 5, A–D). Each Ab titer profoundly increased within 5 to 7 days of MBP p68–86/CFA immunization (p < 0.001 compared with day 0). The increase was simultaneous with the accelerated transcription of each chemokine mRNA at the site of inflammation (Fig. 1), suggesting that generation of each gene product at the site of inflammation elicits the production of anti-self Abs. Each Ab titer peaked after the onset of disease (day 10–12) and returned to background within 40 days. Our data therefore clearly show that transcriptional up-regulation of individual chemokines in the CNS can provide protection from disease progression.

Finally, the competence of C-C chemokine naked DNA vaccines to render long-lasting protective immunity against EAE was evaluated. Rats were subjected to three weekly injections of C-C chemokine naked DNA vaccines, as described above (Expt. 1, Fig. 2 A). Two months after last vaccine was administered, EAE was actively induced. Rats immunized with either MIP-1α or MCP-1 DNA vaccines were highly protected against EAE (incidence of 0/4 for each treatment, compared with 4/4, with a maximal score of 1.25 ± 0.28 in either control or pcDNA3-treated rats, p < 0.001 for each comparison). MIP-1β naked DNA vaccination, however, aggravated the disease (incidence 4/4 with a maximal score of 2.5 ± 0.33, p < 0.013 for each comparison). Thus, MIP-1α and MCP-1 DNA vaccines generate long-lasting protective immunity against autologous cytokines when such a response is needed.

An ideal way of treating a disease caused by a malfunction of the immune system in distinguishing self from foreign would be by encouraging this system to elicit self-protective immunity, and thus restrain its own harmful reactivity only when such a response is needed. This task has been achieved in the current study using the novel technology of naked DNA vaccination.

We previously have used RT-PCR verified by Southern blotting analysis to follow the trafficking of T cells to the site of inflammation during the course of transferred EAE and distinguished between selective and nonselective stages in leukocyte homing to the CNS (3). Based on these data, we have described the development of EAE as a sequential event in which a primary influx (days 0–2) activates the blood brain barrier to allow accumulation of a secondary influx of endogenous leukocytes and the initiation of the disease (days 5–9) (3). Using the same experimental system and the same strategy, we have now shown a positive correlation in time course between the accumulation of the secondary influx at the site of inflammation (3) and an elevated expression of MIP-1α, MCP-1, and MIP-1β at the site of inflammation (Fig. 1 A). Each of the above C-C chemokines is well known for its competence to attract monocytes and T cells to a site of inflammation and for its ability to elicit the expression of various adhesion molecules that mediate the trafficking of these cells (3). Thus, the positive correlation in time course between chemokine expression and cell accumulation at the target organ may be explicated by the putative biologic functions of these chemokines. Unexpectedly, RANTES transcription augmented in EAE brains only after recovery. While similar results were obtained previously in a murine model of disease (40), the biologic implications of this observation are not fully understood.

MIP-1α or MCP-1 DNA vaccines prevented EAE. MIP-1β naked DNA significantly aggravated the disease, and only the generation of in vivo immune response to RANTES naked DNA had no notable effect on EAE manifestation. Thus, intervention in EAE development by C-C chemokine DNA vaccines was effective only for those chemokines that were highly transcribed during the development of the inflammation. This emphasizes the pivotal role of these chemokines in the pathogenesis of EAE. It is possible, although still to be proved, that RANTES plays a role in the establishment and maintenance of the resistant state following recovery.

DNA vaccines represent a novel means of expressing Ags in vivo for the generation of both humoral and cellular immune responses (10, 14, 41, 42, 43). This technology has proven successful in obtaining immunity not only to foreign Ags and tumors, but also to self Ags, such as TCR V genes (17) or autologous cytokines (42). C-C chemokines were selected as candidates for DNA vaccination mostly because of their well-established role in cell migration to a target organ (22, 23, 44, 45, 46, 47, 48, 49). Since DNA vaccination elicits both cellular and humoral responses against products of a given construct (10, 14, 41, 42, 43), it is difficult to know which of these responses contributed more to the development of EAE resistance in MCP-1 and MIP-1α DNA-vaccinated rats. It has, however, been shown that rabbit anti-MIP-1α Abs were capable of blocking EAE in a murine model (33), and an antagonist of MCP-1 markedly inhibited arthritis in the MRL-lpr mouse (34). Under our experimental conditions, vaccination with MCP-1 DNA elicited a significant cross-reactive immune response to MIP-1α (Fig. 6,B; an Ab titer of 21.25 ± 0.99 to self, 17 ± 0.66 to MIP-1α, and 11 ± 0.47 to either MIP-1β or RANTES). Our data clearly show that anti-chemokine Abs produced by naked DNA vaccination are neutralizing Abs (Table II) and can provide subsequent protection from severe EAE (Fig. 7). Thus, it is conceivable that these Abs contribute to disease inhibition in MIP-1α and MCP-1 naked DNA-vaccinated rats.

A major disadvantage in treating chronic diseases with xenogenic neutralizing Abs lies in their immunogenicity. This has motivated investigators to develop chimeric humanized Abs (reviewed in 50 , and mAbs engineered with human Ig heavy and light chain yeast artificial chromosome (51). However, following repeated immunization, these engineered Abs do trigger an apparently allotypic response. The therapeutic strategy suggested by our study is of advantage over the above methods since it resulted in the generation of immunity to autologous Ag. In addition, our study reveals an unexpected, yet extremely important, advantage in applying C-C chemokine DNA vaccination. It appears that the immune response to each of the given DNA constructs elicited only during the course of disease and only at the time when the transcription of the related chemokine profoundly elicited at the site of inflammation EAE induction (Figs. 1 and 4).

Finally, a recent study shows a coordinated chemokine up-regulation in brain and spinal cord during clinical relapse in mice with relapsing EAE (52). This emphasizes the importance of treating a disease caused by a malfunction of the immune system in distinguishing self from foreign, such as multiple sclerosis, by encouraging this system to elicit anti-self-protective immunity, and thus restrain its own harmful activity only when such a response is needed.

We thank Dr. H. Gershon for creative discussion and for reading the manuscript.

1

This study was supported by Israel Cancer Research Foundation (ICRF), Israel Science Foundation, Israel Ministry of Science and Arts, Israel Ministry of Health, The Dankner Foundation for Medical Research, Chutick Foundation for Brain Research, and Bloom Foundation for Medical Research.

3

Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; CNS, central nervous system; MBP, myelin basic protein; MCP, monocyte-chemotactic protein; MIP, macrophage-inflammatory protein; SCF, spinal cord fluid.

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