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The Journal of Immunology, 2003, 170: 1806-1813.
Copyright © 2003 by The American Association of Immunologists

Suppressive DNA Vaccination in Myelin Oligodendrocyte Glycoprotein Peptide-Induced Experimental Autoimmune Encephalomyelitis Involves a T1-Biased Immune Response 1

Anna Lobell2,*, Robert Weissert{dagger}, Sana Eltayeb*, Katrien L. de Graaf{dagger}, Judit Wefer*, Maria K. Storch{ddagger}, Hans Lassmann{ddagger}, Hans Wigzell§ and Tomas Olsson*

* Neuroimmunology Unit, Center for Molecular Medicine, Karolinska Hospital, Stockholm, Sweden; {dagger} Department of Neurology, University of Tuebingen, Tuebingen, Germany; {ddagger} Neurological Institute, University of Vienna, Vienna, Austria; and § Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vaccination with DNA encoding a myelin basic protein peptide suppresses Lewis rat experimental autoimmune encephalomyelitis (EAE) induced with the same peptide. Additional myelin proteins, such as myelin oligodendrocyte glycoprotein (MOG), may be important in multiple sclerosis. Here we demonstrate that DNA vaccination also suppresses MOG peptide-induced EAE. MOG91–108 is encephalitogenic in DA rats and MHC-congenic LEW.1AV1 (RT1av1) and LEW.1N (RT1n) rats. We examined the effects of DNA vaccines encoding MOG91–108 in tandem, with or without targeting of the hybrid gene product to IgG. In all investigated rat strains DNA vaccination suppressed clinical signs of EAE. There was no requirement for targeting the gene product to IgG, but T1-promoting CpG DNA motifs in the plasmid backbone of the construct were necessary for efficient DNA vaccination, similar to the case in DNA vaccination in myelin basic protein-induced EAE. We failed to detect any effects on ex vivo MOG-peptide-induced IFN-{gamma}, TNF-{alpha}, IL-6, IL-4, IL-10, and brain-derived neurotropic factor expression in splenocytes or CNS-derived lymphocytes. In CNS-derived lymphocytes, Fas ligand expression was down-regulated in DNA-vaccinated rats compared with controls. However, MOG-specific IgG2b responses were enhanced after DNA vaccination. The enhanced IgG2b responses together with the requirement for CpG DNA motifs in the vaccine suggest a protective mechanism involving induction of a T1-biased immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Deoxyribonucleic acid vaccination can induce Ag-specific CD4+, CD8+ and CTL responses that are protective against infectious disease (1, 2). DNA vaccination can also protect from autoimmune disease (3, 4, 5, 6, 7, 8), although there are a few reports of exacerbation of autoimmunity after DNA vaccination (9, 10, 11, 12). We have previously reported that vaccination with DNA encoding the autoantigenic myelin basic protein (MBP)3 peptide 68–85 targeted to IgG suppresses experimental autoimmune encephalomyelitis (EAE) actively induced with the same peptide (4, 5, 13). EAE is an animal model of multiple sclerosis (MS) and can be induced in rodents by immunization with myelin proteins or peptides, such as MBP, proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) (14). In MBP-EAE, the protective effect of DNA vaccination was dependent on 1) expression of the hybrid gene encoding MBP68–85 (4), 2) targeting of the gene product to IgG (4), and 3) immunostimulatory CpG DNA motifs of the plasmid DNA backbone (5). The effect was strictly Ag specific (13). Furthermore, the suppressive effect of the DNA vaccine was inhibited by coinjection of DNA encoding IL-4, IL-10, or TNF-{alpha} (5). There was no correlation between the protective capability of the DNA vaccine and the ex vivo MBP peptide-induced T cell responses, and there were no signs of a T2-biased immune response (4, 5, 13).

Protective effects of DNA vaccination in EAE are not restricted to MBP. Vaccination with a construct encoding PLP peptide139–151 in SJL/J mice suppressed PLP139–151-induced EAE (6). In that report a mechanistic explanation was suggested, in that DNA vaccination altered the frequency of expression of CD80 and CD86 on APCs in the spleen. A combination of PLP139–151-encoding DNA vaccine and IL-4-encoding DNA suppressed subsequently induced EAE (8). In those experiments protection correlated to the induction of T2 immunity.

The phenomenon of epitope spreading in EAE implies that the disease-promoting immune response may change to new epitopes within the same Ag or to other myelin proteins with time (15, 16, 17). In MS, several myelin autoantigens are also recognized (18), and there is evidence suggesting epitope spreading in human disease (16). In such situations a potential protective DNA vaccine, due to its high Ag specificity (13), would need to act on several epitopes. We considered it likely that requirements for DNA vaccination effects may differ depending on the targeted autoantigen. We therefore extended our previous studies to include DNA vaccination with a second important myelin autoantigen with a putatively different pathogenic action, MOG91–108. MOG91–108 is dominantly encephalitogenic in the inbred DA (RT1av1) and the MHC congenic Lewis rat strains LEW.1AV1 (RT1av1) and LEW.1N (RT1n) (19).

We constructed DNA vaccines encoding MOG91–108 in tandem, either with or without fusion to an IgG-binding synthetic analog of staphylococcal protein A, ZZ (20). Fusion of the autoantigen to ZZ was essential for the protective effect in the MBP-EAE system (4). LEW.1AV1, LEW.1N, and DA rats were vaccinated with DNA before induction of EAE with MOG91–108 in CFA. Effects on 1) clinical and histopathological signs of EAE and 2) Ag-specific T and B cell responses were studied. Vaccination with DNA encoding MOG91–108 suppressed clinical signs of EAE. There were different requirements for protection against EAE in MBP- vs MOG-induced EAE, since the MOG peptide vaccine lacking ZZ was effective, whereas targeting to IgG made the vaccine nonsuppressive. However, similar to DNA vaccination in MBP-induced EAE, CpG motifs were required in the plasmid backbone for efficient DNA vaccination. Vaccinated vs control rats did not differ in their ex vivo T cell responses, IFN-{gamma} secretion, or IFN-{gamma} transcription in splenocytes or CNS-derived lymphocytes. However, MOG-specific IgG2b Abs were increased in protected animals. This together with the requirement for CpG DNA suggest an enhanced type 1-like response in the protected rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides, Ag, and mitogen

Peptides SDEGGYTCFFRDHSYQEE from rat sequence MOG91–108 and HYGSLPQKSQRSQDENPV from guinea pig sequence MBP68–85 were synthesized using the F-moc/2-(1-H-benzotiazol-1-yl)1,1,3,3,-tetramethyluronium hexafluorophosphate strategy (A. Engstrom, University of Uppsala, Uppsala, Sweden). Recombinant rat MOG (rMOG; extracellular domain; aa 1–125) was produced in Escherichia coli as previously described (21). Con A was purchased from Sigma-Aldrich (St. Louis, MO).

Plasmid construction

A summary of the different DNA vaccines and controls is presented in Table I.


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  		Table I. Properties of DNA vaccines used in experimentsa

 
pZZ/MOG91–108. A 94-bp murine heavy chain IgG signal sequence (ss) was ligated upstream and in-frame of a 385-bp fragment encoding ZZ, a dimerized synthetic analog to staphylococcal protein A (20). Downstream of the coding sequence of ZZ, seven AvaI-AvaI fragments encoding MOG91–108 were ligated in-frame (Fig. 1A).



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  		FIGURE 1. A, Plasmid map of DNA vaccine pZZ/MOG91–108. Seven repeats of oligonucleotides encoding autoantigen MOG91–108 were cloned downstream of a murine ss fused to a dimerized synthetic analog of the IgG-binding B domain of staphylococcal protein A gene, ZZ. As a negative control, a corresponding plasmid coding for ZZ was constructed (pZZ). B, Plasmid map of DNA vaccine pMOG91–108. Seven repeats of oligonucleotides encoding autoantigen MOG91–108 were cloned downstream of a murine ss. As a negative control, pCI vector was used.

 
pMOG91–108. A linker containing AvaI and BbsI sites was ligated downstream and in-frame of the ss fragment. Downstream of the ss fragment, seven AvaI-AvaI MOG91–108 fragments were ligated in-frame (Fig. 1B). The ss/ZZ/MOG91–108 and the ss/MOG91–108 fragments were cloned into the eukaryotic expression vector pCI (Promega, Madison, WI).

pZZ. An ss/ZZ fragment containing ss and ZZ in-frame was cloned into pCI.

pK0. Construction of pK0 has previously been described (5). pK0 is a eukaryotic expression vector that lacks immunostimulatory CpG DNA motifs of AACGTT sequence.

pK0-MOG91–108. The ss/MOG91–108 fragment was cloned into pK0 vector.

pK3. Construction of pK3 has previously been described (5). Briefly, a linker containing three AACGTT CpG DNA motifs was inserted into pK0. pK0 and pK3 are thus identical, except for the three CpG motifs.

pK3-MOG91–108. The ss/MOG91–108 fragment was cloned into pK3 vector.

The E. coli host was XL1-Blue or XL10-Gold (all pK0 and pK3 constructs; Stratagene, La Jolla, CA).

DNA oligomer synthesis

Single-stranded oligomer DNA coding for three CpG DNA motifs was synthesized at Cybergene (Huddinge, Sweden) and diluted in water. The DNA sequence is 5'-TTGGAACGTTCCTTTCCAACGTTGGTTTGGAACGTTCCTT-3'. CpG DNA motifs are underlined.

Plasmid preparation

Plasmid DNA was prepared by the Qiagen plasmid preparation protocol. Endotoxins were removed in an additional step (Endofree buffer set; Qiagen, Santa Clarita, CA).

Plasmid DNA injections and cardiotoxin pretreatment

Four- to 5-wk-old LEW.1AV1 (RT1av1; Charles River Laboratories, Uppsala, Sweden), LEW.1N (RT1n), and DA (B&K, Stockholm, Sweden) female rats were used in the experiments. LEW.1N were originally obtained from Prof. H. Hedrich (Medizinische Hochschule, Hannover, Germany) and were then locally bred. Rats were injected with 100 µl of 10 µM cardiotoxin (Latoxan, Rosans, France) into the gastrocnemii muscles. Seven days later, rats were injected with 800 µg of DNA at 2.0 mg/ml in PBS, divided into four 100-µl injections administered in the tibialii and gastrocnemii muscles, of pZZ/MOG91–108, pZZ, pMOG91–108, pCI, pK0-MOG91–108, pK0, pK3-MOG91–108, or pK3. In the last experiment 100 µg of CpG DNA was added to pMOG91–108 or pCI before i.m. injection.

EAE induction and clinical evaluation

Three to 4 wk after DNA vaccination, rats were injected intradermally at the base of the tail with 200 µl of inoculum containing a 1/1 dilution of 100 µg of MOG91–108 in PBS emulsified in CFA, consisting of IFA (Sigma-Aldrich) and 0.5 mg of heat-inactivated Mycobacterium tuberculosis (H37 RA strain; Difco, Detroit, MI). Rats were clinically scored and weighed daily. The symptoms were scored as follows: grade 1, tail weakness or tail paralysis; grade 2, hind leg paraparesis; grade 3, hind leg paralysis; and grade 4, complete paralysis (tetraplegy), moribund state, or death. Weight loss was calculated as the difference between the weight on the day of induction of disease compared with the lowest weight during disease, divided by the weight on the day of induction of disease.

Determination of MBP68–85-specific IgG and IgG isotype responses

ELISA plates were coated with 10 µg/ml MOG91–108 or 2 µg/ml rMOG in carbonate buffer, pH 9.6. Rat sera were diluted 1/50 in 5% milk powder and 0.02% Tween 20 in PBS. Wells were incubated 2 h with sera, washed in 0.02% Tween 20 in PBS, and incubated for 2 h with a 1/1000 dilution of alkaline phosphatase-conjugated goat anti-rat IgG (BioSource, Camarillo, CA), or monoclonal alkaline phosphatase-conjugated mouse-anti rat IgG1, IgG2a, or IgG2b (BD PharMingen, San Diego, CA), respectively, in 5% milk powder and 0.02% Tween 20 in PBS. p-Nitrophenyl phosphate (Sigma-Aldrich) was used as substrate, and absorbance was read at 405 nm.

Cell preparation and culture

Spleens were disrupted, and cells were suspended in DMEM (Life Technologies, Gaithersburg, MD). Erythrocytes were lysed for 5 min with lysis buffer containing 0.84% NH4Cl. Mononuclear cells were resuspended in complete medium containing DMEM supplemented with 5% heat-inactivated newborn calf serum (Life Technologies), 1% penicillin/streptomycin (Life Technologies), and 50 µM 2-ME (Life Technologies) and flushed through a 70-µm pore size plastic strainer (BD Biosciences, Mountain View, CA), adjusted to 2 x 106 cells/ml, and cultured at 37 C in a humidified atmosphere containing 5% CO2.

Preparation of CNS-derived lymphocytes

Preparation of CNS-derived lymphocytes using Percoll separation has been described previously (22). In short, killed rats were perfused with 50 ml of PBS, and rat CNS was dissected out and homogenized in Percoll/buffer solution. CNS homogenate was centrifuged in a 30–63% Percoll gradient, and lymphocytes were isolated, washed, and frozen at –70 C.

ELISA to access cytokine production ex vivo

ELISA kits for detection of secreted rat IFN-{gamma}, IL-4, IL-10, and monocyte chemotactic protein-1 (MCP-1) were purchased from BioSource. Supernatants from mononuclear cells, which had been incubated for 48 h at a concentration of 2 x 106 cells/ml with or without MOG91–108, MBP68–85, or the mitogen Con A, were analyzed. The procedure was performed as recommended by the manufacturer.

RT-PCR and quantification of cytokine, neurotropin, and Fas ligand (FasL) mRNA levels

RT-PCR and real-time PCR to quantify levels of rat IFN-{gamma}, TNF-{alpha}, IL-10, IL-6, brain-derived neurotropic factor (BDNF), and FasL have been described previously (22, 23). Briefly, 4 x 106 splenocytes were stimulated with or without MOG91–108 for 24 h. RT-PCR was performed as previously described. RNA was prepared directly from CNS-derived lymphocytes without any in vitro stimulation. Amplification was performed using an ABI PRISM 7700 Sequence Detection System (Perkin-Elmer, Norwalk, CT). The relative amounts of the endogenous control GAPDH mRNA and target mRNA in each sample could be deduced from the GAPDH and target mRNA standard curves, respectively. The amount of mRNA in each sample was calculated as the ratio between the relative amount of cytokine/neurotropin/FasL and the relative amount of the corresponding endogenous control, GAPDH mRNA.

Histopathological evaluation

Histopathological examinations were performed as previously described (24).

Statistics

Differences in clinical EAE score or T and B cell reactivity between groups were tested with the Mann-Whitney U test. Differences in mortality between groups were tested with Fisher’s exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Vaccination with DNA encoding MOG91–108 reduces clinical signs of EAE and mortality

We first constructed DNA vaccines encoding MOG91–108 in tandem, with or without fusion to IgG-binding ZZ (Table I and Fig. 1). We wanted to determine whether the requirements differed for suppressive DNA vaccination in MOG peptide-induced EAE compared with MBP68–85-induced EAE (4). Rats were injected with cardiotoxin 7 days before DNA vaccination. Three to 4 wk after DNA vaccination, EAE was induced with MOG91–108 in CFA. A summary of the effects of DNA vaccination on clinical and histopathological signs of EAE is presented in Table II.


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  		Table II. Effect of vaccination with DNA encoding MOG91–108 on clinical and histopathological signs of EAE

 
Vaccination with pZZ/MOG91–108 (Table I and Fig. 1A) had no effect on the mean maximum clinical EAE score in either LEW.1AV1 (p = 0.95) or LEW.1N (p = 0.64) rats (Table II and Fig. 2, A and B), compared with pZZ-treated rats, but the mean accumulated EAE score was mildly reduced in LEW.1AV1 (p = 0.05) rats. However, vaccination with pMOG91–108 (Table I and Fig. 1B) reduced the mean maximum (p = 0.03) and mean accumulated (p = 0.05) EAE scores in LEW.1AV1 rats compared with those in pCI-treated controls (Table II and Fig. 2C). A similar result was obtained in LEW.1N rats (Table II and Fig. 2D). The mean accumulated EAE score was significantly reduced in the pMOG91–108-treated LEW.1N rats (p = 0.049), and the mean maximum score was also reduced (p = 0.06). Three rats had to be excluded from the analysis of the mean accumulated EAE score because they died between days 12–13, causing bias of the mean accumulated value. There was a dramatic reduction in mortality in pMOG91–108-treated LEW.1N rats compared with controls; four of seven rats died in the pCI-treated group, compared with none in the pMOG91–108-treated group (p = 0.02). Mortality in the pMOG91–108-treated LEW.1AV1 rats was also reduced (not significant). Untreated LEW.1AV1 rats were immunized with MOG91–108 in parallel with the treated rats to exclude that pCI treatment had any effect on the clinical outcome of the treatment (Table II). Vaccination with pMOG91–108 was highly protective in DA rats; the mean maximum (p = 0.03) and mean accumulated (p = 0.009) EAE scores were strongly reduced compared with those in pCI-treated controls (Table II and Fig. 2E). The percent weight loss was also measured for all rats. The mean weight loss was reduced in the pMOG91–108-vaccinated LEW.1AV1 (p = 0.02; Fig. 3A), LEW.1N (p = 0.07; Fig. 3B), and DA rats (p = 0.03; Fig. 3C) compared with controls, and the data correlated well with the accumulated EAE score.



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  		FIGURE 2. Vaccination with DNA encoding MOG91–108 suppresses MOG91–108-induced EAE in three different rat strains, sharing either genetic background (LEW.1AV1 and LEW.1N) or MHC (LEW.1AV1 and DA). Only DNA vaccine lacking ZZ was efficient. The mean clinical EAE score ± SEM after vaccination with pZZ/MOG91–108 or pZZ in LEW.1AV1 rats (A) or LEW.1N rats (B) is shown. In the second row, the mean clinical EAE score after vaccination with pMOG91–108 or pCI in LEW.1AV1 rats (C), LEW.1N rats (D), or DA rats (E) is shown. Rats were injected once with DNA vaccine or control 3–4 wk before induction of EAE with MOG91–108.

 


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  		FIGURE 3. Weight loss was reduced in the pMOG91–108-treated groups compared with controls. The mean percent weight loss ± SEM for pMOG91–108-, pZZ-, pMOG91–108-, or pCI-treated LEW.1AV1 rats (A), LEW.1N rats (B), and pMOG91–108- or pCI-treated DA rats (C). Rats were injected once with DNA vaccine or control 3–4 wk before induction of EAE with MOG91–108.

 
Thus, the requirements differed between DNA vaccination for MBP peptide-induced EAE and MOG peptide-induced EAE, because the ZZ gene was not necessary for the protective effect to occur in the present study, but, instead, made the vaccine inefficient.

CpG DNA content of the vaccine is essential for efficient DNA vaccination

We then investigated the adjuvantic properties of the plasmid backbone, since T1-promoting, noncoding CpG DNA was necessary for efficient DNA vaccination in MBP peptide-induced EAE (5). To compare CpG-deficient and CpG-containing vaccines, we constructed two new DNA vaccines, one containing three CpG AACGTT sequences, pK3-MOG91–108 (Table I), and one lacking AACGTT sequences, pK0-MOG91–108. These two vaccines were identical except for a linker containing three AACGTT sequences. These DNA vaccines and controls were injected into LEW.1AV1 rats before induction of EAE with MOG91–108. Treatment with pK3-MOG91–108 reduced the mean accumulated EAE score by 52% (p = 0.02; Table II and Fig. 4A), while treatment with the CpG DNA-deficient vaccine pK0-MOG91–108 failed to suppress clinical symptoms of EAE (p = 0.86; Table II and Fig. 4B). To determine whether an even higher dose of CpG DNA could abrogate the effect of the DNA vaccination, oligomer CpG DNA containing three AACGTT motifs was synthesized. LEW.1AV1 rats were treated with pMOG91–108 or pCI without oligomer CpG DNA (Fig. 5A). In parallel, rats were treated with 100 µg of CpG DNA added to pMOG91–108 or pCI (Fig. 5B). Three weeks later all rats were immunized with MOG91–108 in CFA. Addition of 100 µg of CpG DNA to the DNA vaccine or control did not alter the clinical outcome of the DNA vaccination (Fig. 5, A and B) compared with that in the pMOG91–108-treated rats. Instead, the mean maximum EAE score was reduced to the same extent in Fig. 5, A (p = 0.04) and B (p = 0.04). Thus, the requirement for CpG DNA in the plasmid backbone was similar for suppressive vaccination with DNA encoding MOG91–108 and DNA encoding MBP68–85.



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  		FIGURE 4. Immunostimulatory CpG DNA motifs are necessary for suppression of EAE after vaccination with DNA encoding MOG91–108 3–4 wk before induction of EAE with MOG91–108 in CFA. The mean clinical EAE score ± SEM after vaccination with pK3-MOG91–108 or pK3 that contain three CpG DNA motifs (A) and CpG DNA-deficient pK0-MOG91–108 or pK0 (B) is shown. Rats were injected once with DNA vaccine or control 3–4 wk before induction of EAE with MOG91–108.

 


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  		FIGURE 5. Addition of oligomer CpG DNA to DNA vaccine or control does not alter the suppressive capability of the DNA vaccination. One hundred micrograms of oligomer DNA coding for three immunostimulatory CpG motifs was added to pMOG91–108 or pCI before injection into rats. The high dose of CpG DNA did not alter the clinical outcome of the DNA vaccination (B) compared with DNA vaccination without addition of oligomer CpG DNA (A). The mean clinical EAE score ± SEM are shown. Rats were injected once with DNA vaccine or control 3–4 wk before induction of EAE with MOG91–108.

 
MOG91–108-specific T and B cell responses after DNA vaccination

We measured MOG91–108-induced immune responses in the form of ex vivo cytokine/chemokine secretion and mRNA transcription, searching for putative mechanisms involved in the suppression of MOG91–108-induced EAE in LEW.1AV1 after vaccination with pMOG91–108 or pK3-MOG91–108. Firstly, cytokine/chemokine secretion was tested, and IFN-{gamma}, IL-4, and IL-10 were selected due to their defined roles in T1/T2 bias. In addition, MCP-1 production was studied, since MCP-1 can regulate oral tolerance induction in EAE (25) and acts as an anti-inflammatory chemokine in EAE (26). We measured Ag-induced IFN-{gamma} and MCP-1 in vitro responses of splenocytes from MOG91–108-immunized and pMOG91–108- or pCI-treated LEW.1AV1 rats 12 days after immunization. The IFN-{gamma} production of splenocytes from pMOG91–108- and pCI-treated rats did not differ (Fig. 6A). Likewise, MCP-1 production was the same in splenocytes from pMOG91–108- and pCI-treated rats (Fig. 6B). Production of IL-4 and IL-10 in response to MOG91–108 was undetectable in all pMOG91–108-treated rats and controls (data not included). IL-10, but not IL-4, was produced in equal amounts in response to Con A in both groups (data not included).



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  		FIGURE 6. Suppressive DNA vaccination does not alter Ag-specific IFN-{gamma} secretion from splenocytes during the acute phase of EAE. The mean ± SEM (A) IFN-{gamma} and (B) MCP-1 secretion at 12 days after immunization is shown. LEW.1AV1 rats were treated with pMOG91–108 or pCI 3–4 wk before immunization with MOG91–108 in CFA. Supernatants from mononuclear cells exposed in vitro to MOG91–108, MBP68–85, or Con A for 48 h were tested in cytokine ELISA for the presence of IFN-{gamma} and MCP-1, respectively.

 
Secondly, we measured Ag-induced mRNA transcription of IFN-{gamma}, TNF-{alpha}, IL-6, IL-10, BDNF, and FasL in splenocytes from MOG91–108-immunized and pK3-MOG91–108-, pK3-, pK0-MOG91–108-, or pK3-treated LEW.1AV1 rats 12 days after immunization. TNF-{alpha} and IL-6 are proinflammatory cytokines, and BDNF was studied because it can be neuroprotective (23). FasL transcription was studied because it is transcribed in T cells that are undergoing apoptosis (27). IFN-{gamma} transcription (Fig. 7A) did not differ between the groups (p = 0.11). We failed to detect any significant differences in transcription of IL-6 (Fig. 7B), IL-10 (Fig. 7C), TNF-{alpha} (Fig. 7D), BDNF (Fig. 7E), and FasL (Fig. 7F) between the groups that correlated with the suppressive capability of pK3-MOG91–108. Next, we measured mRNA transcription of IFN-{gamma}, TNF-{alpha}, IL-6, IL-10, BDNF, and FasL of CNS-derived lymphocytes from MOG91–108-immunized and pMOG91–108- or pCI-treated LEW.1AV1 rats 11 days after immunization. We failed to detect any significant differences in transcription of IFN-{gamma}, IL-6, IL-10, TNF-{alpha}, or BDNF (Fig. 8). However, FasL transcription was higher (p = 0.02) in the control group than in the pMOG91–108-treated group (Fig. 8) Thus, we failed to detect any signs of induction of T1/T2-biased cytokine production after suppressive DNA vaccination and EAE production.



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  		FIGURE 7. The mean ± SEM transcription of IFN-{gamma} (A), IL-6 (B), IL-10 (C), TNF-{alpha} (D), BDNF (E), and FasL (F) in splenocytes stimulated with MOG91–108 do not correlate with the suppressive capability of the DNA constructs. On day 12 after immunization, splenocytes from MOG91–108-immunized and DNA-vaccinated rats were cultured 24 h with or without Ag. The amount of mRNA in each sample was calculated as the ratio between the relative amount of cytokine/neurotropin/FasL mRNA and the relative amount of the corresponding endogenous control, GAPDH mRNA.

 


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  		FIGURE 8. The mean transcription of IFN-{gamma}, IL-6, IL-10 TNF-{alpha}, and BDNF mRNA is similar in CNS-derived lymphocytes from pMOG91–108-vaccinated and pCI-treated LEW.1AV1 rats. Transcription of FasL mRNA is higher (p < 0.02) in the control group than in the DNA-vaccinated group. On day 11 after immunization, CNS-derived lymphocytes were prepared using Percoll separation. The amount of mRNA in each sample was calculated as the ratio between the relative amount of cytokine/neurotropin/FasL mRNA and the relative amount of the corresponding endogenous control, GAPDH mRNA. The horizontal line represents the mean ratio. **, p < 0.02.

 
Next we measured MOG91–108-specific IgG and IgG isotype responses from MOG91–108-immunized and DNA-vaccinated LEW.1AV1 rats 12 days after immunization to study the profile of B cell reactivity to MOG91–108. The total Ag-specific IgG responses did not differ between pZZ/MOG91–108-treated (Fig. 9A) or pMOG91–108-treated (Fig. 9B) rats and controls. IgG1 responses were almost undetectable in both groups, but the IgG2a and IgG2b responses were enhanced in the pMOG91–108-treated group compared with controls (Fig. 9B). The results were similar in experiment 3 (data not included). We then studied the profile of B cell reactivity to rMOG. We measured MOG-specific IgG and IgG isotype responses from MOG91–108-immunized and DNA-vaccinated LEW.1AV1 rats 12 days after immunization. The Ag-specific IgG levels did not differ between the pZZ/MOG91–108-treated group and controls (Fig. 9C), but were higher in the pMOG91–108-treated group compared with controls (Fig. 9D). Again, IgG1 levels were almost undetectable, whereas IgG2b levels were higher in the pMOG91–108-treated group compared with controls (Fig. 9D). IgG and IgG isotype responses in the LEW.1N rats were similar to the responses in the LEW.1AV1 rats (data not included).



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  		FIGURE 9. Ag-specific IgG2b Ab production is enhanced in rats treated with the suppressive DNA vaccine pMOG91–108. A and B, Mean anti-MOG91–108-specific IgG and IgG isotype responses at a 1/50 serum dilution. LEW.1AV1 rats received pZZ/MOG91–108 or pZZ (A) or pMOG91–108 or pCI (B) 3–4 wk before induction of EAE with MOG91–108 in CFA. Rat sera were collected from rats 12 days after immunization. **, p < 0.04. C and D, Mean anti-rMOG-specific IgG and IgG isotype responses at a 1/50 serum dilution. LEW.1AV1 rats received pZZ/MOG91–108 or pZZ (C) or pMOG91–108 or pCI (D) 3–4 wk before induction of EAE with MOG91–108 in CFA. Rat sera were collected from rats 12 days after immunization. **, p < 0.04.

 
Histopathology

It was possible to conduct a limited histopathological evaluation of certain cohorts in this study. The severe disease course in LEW.1N precluded an accurate histological comparison between the groups (21). In LEW.1AV1 rats the degree of inflammation or demyelination in the CNS did not differ between the groups during the acute phase of the disease (Table II). However, on day 27 after immunization with MOG peptide, the degree of inflammation in the CNS tended to be reduced in the pMOG91–108-treated group compared with controls (p = 0.055).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We here demonstrate that DNA vaccination can protect against an important encephalitogenic myelin protein, MOG. Thus the principle of DNA vaccination is applicable for all three main encephalitogens MBP, PLP and MOG, implicated in the pathogenesis of MS. Accordingly, if a disease is driven by several epitopes from these autoantigens a combinatorial vaccine could theoretically be constructed. Interestingly, a combinatorial approach delivering an MBP- plus a MOG-peptide was shown to be effective in mice (28).

We also demonstrate a similar vaccination effect in two different MHC haplotypes and in rats with two different genetic backgrounds that share the same MHC, a finding of interest in view of the nonexclusive MHC restriction of human MS (29). Only the DNA vaccine lacking the ZZ gene was efficient in MOG peptide-induced EAE in all rat strains tested.

Interestingly, one aspect of the vaccine construct requirements to obtain a protective effect differed between the MOG-EAE and MBP-EAE systems, while another was similar in the two systems. 1) As mentioned above, in contrast to the MBP-EAE system, only the DNA vaccine lacking ZZ was protective in MOG-EAE. We have no direct experimental data explaining this. However, it is probably due to differences in immune pathogenesis between these two Ags. MBP-induced EAE is considered a pure T1-driven disease (30). Encephalitogenicity in MOG-induced EAE appears more complex and possibly includes a direct action of pathogenic Abs (19, 21, 31). Thus, the requirements for immunoselective prophylaxis and treatment might be different. The construct encoding ZZ will target the gene product to IgG and theoretically to Fc{gamma}R-bearing cells, including all professional APCs and NK cells. To our knowledge, whether ZZ blocks binding of Fc to Fc{gamma}R has not been studied, but the two molecules, Fc{gamma}R and ZZ, bind to Fc at different positions (32, 33), suggesting that ZZ does not block Fc-Fc{gamma}R binding. Engagement of Fc{gamma}R in EAE can act on at least two levels of EAE induction. 1) Fc{gamma}R can affect Ag presentation, because APCs can take up Ag through Fc{gamma}R (34). Macrophages express Fc{gamma}R more readily than dendritic cells (34). Targeting the gene product to IgG may skew the Ag uptake toward macrophages, while uptake of the soluble gene product may be directed toward dendritic cells. 2) Fc{gamma}R may mediate local tissue damage in the CNS, since Fc{gamma}R-deficient mice were protected from EAE, although they had equal amounts of circulating MOG-specific T and B cells (35). We consider it more likely that by targeting the gene product to IgG we affected Ag uptake during initiation of the immune response, because we DNA vaccinated the rats weeks before EAE induction. 3) In contrast, CpG DNA in the plasmid backbone of the DNA vaccine was required for protection in both MOG and MBP systems (5).

The mechanism mediating the protective effect remains unclear. The requirement for CpG DNA in the DNA vaccine argues for an activated APC function and against induction of T2 immunity. Unmethylated CpG DNA binds to Toll-like receptor-9 (TLR-9) on APCs (36). Binding of CpG DNA to TLR-9 leads to cellular activation, with production of T1 cytokines IL-12 and IL-18, expression of costimulatory molecules, and increased Ag presentation (36). During EAE induction with myelin peptide in CFA, receptors other than TLR-9 will be involved; e.g., TLR-4 recognizes LPS, and TLR-2 recognizes lipoproteins from M. tuberculosis. Thus, the quality of the T cell response may be skewed during EAE induction if APCs have already been activated through TLR-9 upon DNA vaccination. Nevertheless, we observed no increase or decrease in Ag-induced IFN-{gamma} production in splenocytes or CNS-derived lymphocytes. However, in CNS-derived lymphocytes from pCI-treated LEW.1AV1 rats, we observed increased FasL expression in rats compared with cells from DNA-vaccinated rats. Because rats in the control group are more severely ill, we hypothesize that T cells will attempt to down-regulate ongoing CNS inflammation by, for instance, expressing FasL during the peak of disease. In the DNA-vaccinated, protected rats neuroinflammation is not severe. Instead of a T1/T2 shift, regulatory T cells may have been induced, or the protective effect could be mediated through cells other than T cells (e.g., NK cells), since NK cells are activated by IL-12 (36).

The absence of Ag-specific IL-4 and IL-10 secretion argues against a T2 immune deviation, and the extremely low levels of IgG1 are consistent with this. This particular peptide does not promote T2-driven IgG1 production in the rat strains used. Such low rMOG-specific IgG1 levels have also previously been observed in the LEW.1A (RT1a) rat strain, which is closely related to the LEW.1AV1 (RT1av1) strain (data not included), but not in LEW (RT1l), excluding technical difficulties in the detection of this IgG isotype as an explanation for this negative finding. Furthermore, preliminary data from our laboratory suggest that Ag-specific IFN-{gamma}, but not IL-10, is induced after DNA vaccination, but before EAE production, with pMOG91–108, but not with pCI.

Bourquin et al. (11) reported exacerbation of MOG-induced EAE and enhanced production of pathogenic Abs after vaccination with DNA encoding MOG in SJL mice. Here we did not assess the pathogenicity of Abs, although we observed enhanced Ag-specific IgG2b production in the DNA-vaccinated group during the peak of disease. IgG2b is considered to be T1 driven, and this finding is consistent with the requirement for CpG DNA in our system. Garren et al. (8) report protection from ongoing EAE if they covaccinate mice with a DNA vaccine encoding MOG plus IL-4-encoding plasmid DNA with associated induction of T2 immunity. Covaccination with DNA encoding PLP peptide plus IL-4-coding DNA suppressed subsequently induced EAE. However, vaccination with DNA encoding PLP peptide was suppressive even without covaccination with IL-4-coding DNA (6). A T2 shift was therefore not necessary for DNA vaccination to be efficient in that experimental system.

The requirement for CpG DNA, the enhanced production of Ag-specific IgG2b Abs, the low levels of IL-4 and IL-10, and Ag-specific IgG1 Abs argue for an induction of protective T1-like immunity in our system. Further studies of the exact molecular mechanism(s) underlying the observed protection from EAE are warranted and ongoing in our laboratory. Such studies are necessary for prediction of the safety and efficacy of DNA vaccination in human organ-specific autoimmune diseases. Furthermore, data on the protective mechanism may reveal new drug target candidates for intervention in human autoimmune diseases.


   Acknowledgments
 
We thank Assoc. Prof. Robert Harris for critical reading of the manuscript, and Britt Dahlgren for excellent technical assistance.


   Footnotes
 
1 This work was supported by European Community Biomed2 (BMH4-97-2027), Swedish Medical Research Council, Deutsche Forschungsgemeinschaft (1947/2-3), Swedish Foundation for Strategic Research, Swedish Association of Neurologically Disabled, The Tore Nilson Fund, Swedish Cancer Society, SBL vaccin AB, and Pharmacia & Upjohn. Back

2 Address correspondence and reprint requests to Dr. Anna Lobell, Neuroimmunology Unit, Center for Molecular Medicine, L8:04, Karolinska Hospital, 171 76 Stockholm, Sweden. E-mail address: anna.lobell{at}cmm.ki.se Back

3 Abbreviations used in this paper: MBP, myelin basic protein; BDNF, brain-derived neurotropic factor; EAE, experimental autoimmune encephalomyelitis; FasL, Fas ligand; MCP-1, monocyte chemotactic protein-1; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; PLP, proteolipid protein; rMOG, recombinant rat MOG; ss, signal sequence; TLR, Toll-like receptor. Back

Received for publication June 3, 2002. Accepted for publication December 9, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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