DNA-based vaccines for multiple sclerosis: Current status and future directions

Clinical Immunology (2012) 142, 76–83

available at www.sciencedirect.com

Clinical Immunology www.elsevier.com/locate/yclim

REVIEW

DNA-based vaccines for multiple sclerosis: Current status and future directions Nicolas Fissolo, Xavier Montalban, Manuel Comabella ⁎ Centre d'Esclerosi Múltiple de Catalunya, CEM-Cat, Unitat de Neuroimmunologia Clínica, Hospital Universitari Vall d'Hebron (HUVH), Barcelona, Spain

Received 11 November 2010; accepted with revision 17 November 2010 Available online 15 December 2010

KEYWORDS Multiple sclerosis; DNA vaccines; Tolerance

Abstract Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that mainly affects young adults between 20 and 40 years of age and leads to significant disability. Although MS is considered to be an immune-mediated disorder, current immunosuppressive therapies fail to inhibit disease progression, and some of them are associated with serious adverse reactions. DNA vaccination is a strategy of immunization based on the injection of genes encoding for target proteins. Depending on the route as well as the dosage of administration, exposure to certain molecules may either stimulate effector responses or induce immune tolerance. A large body of data from the animal model, experimental autoimmune encephalomyelitis (EAE), has demonstrated efficacy of DNA vaccination at inhibiting the disease through the induction of basic tolerizing mechanisms such as anergy, clonal deletion, immune deviation, or induction of regulatory cells. Interestingly, recent phase I and II clinical trials in MS with DNA vaccines have shown positive results in reducing MRI-measured disease activity in patients with relapse-onset MS, and inducing antigen-specific tolerance to myelin-specific B and T cells. Thus, DNA vaccines represent a promising therapeutic approach for MS which also seem to overcome the safety concerns raised by other currently tested therapeutic strategies. Here, we will review existing data from MS and EAE studies on DNA vaccination and discuss on further optimization of the DNA technology in order to improve treatment efficacy. © 2010 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 DNA plasmid vaccines: overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

⁎ Corresponding author. Unitat de Neuroimmunologia Clínica, CEM-Cat, Edif. EUI 2ª planta, Hospital Universitari Vall d'Hebron, Pg. Vall d'Hebron 119-129 08035 Barcelona, Spain. Fax: +34 932746084. E-mail address: [email protected] (M. Comabella). 1521-6616/$ - see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2010.11.011

DNA-based vaccines for multiple sclerosis: Current status and future directions 2.1. Mechanism of action . . . . . . . . . . . . . . . . . . 2.2. Advantages and potential concerns of DNA vaccines 3. Tolerogenic vaccination . . . . . . . . . . . . . . . . . . . . 4. DNA vaccines in EAE . . . . . . . . . . . . . . . . . . . . . . 4.1. Antigen-specific immune modulation . . . . . . . . . 5. Clinical trials of DNA vaccines in MS . . . . . . . . . . . . . 6. Future directions for the use of DNA vaccines in MS . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by perivascular inflammation, demyelination and axonal damage [1]. There are three clinical forms of MS, relapsing remitting MS (RRMS), secondary progressive MS (SPMS) and primary progressive MS (PPMS). Although the etiology of MS remains unknown, there is increasing evidence sustaining the idea that autoimmunity plays a major role in susceptibility to and development of the disease [2]. It has been postulated that the root cause of MS and other autoimmune diseases is an antigen-specific and organ-specific immune attack on the self. Autoreactive immune cells have been isolated from patients with various autoimmune disorders. In MS, several lines of evidence suggested molecular mimicry, bystander activation and epitope spreading as possible mechanisms to initiate and perpetuate disease activity. The largest number of published reports showing the mechanisms triggering inflammation and demyelination of the CNS derives from its animal model, experimental autoimmune encephalomyelitis (EAE). In this model, the disease can be induced by active immunization with components of the myelin sheath, and myelin peptides from the myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) have been identified as immunological antigens [3–6]. Autoreactive T cells against these antigens are essential for the initiation and maintenance of the CNS inflammatory response leading to myelin destruction. Most of the currently used drugs for MS treatment target immune responses, but are not selective for autoreactive T cells. While effective in some cases, this may not offer the ideal strategy to treat the disease since host-protective immune responses could also be altered. Ideally, treatment strategies in MS should aim to selectively restore selftolerance to the autoantigens while leaving the healthy immune system intact. In the present review we summarize existing data from MS and EAE studies supporting the use of antigen-specific therapies based on DNA vaccination to treat MS in a highly selective manner. We also propose future directions for the use of DNA vaccines in MS.

2. DNA plasmid vaccines: overview Conventional vaccines are constructed with components of pathogenic agents. Whole microorganisms (killed or attenuated), tumor cell lysates, antigens and peptides have been used in

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diverse vaccine preparations [7]. However, over the last years, the introduction of antigen-coding genes (DNA vaccines) has represented a new alternative source of vaccine preparations. DNA plasmid vaccines consist of a bacterial plasmid containing a gene encoding the desired antigen. Expression is under the control of a mammalian promoter, a transcription terminator and often an antibiotic resistance gene that allows the selection of the plasmid during production in bacteria [8].

2.1. Mechanism of action Plasmid DNA vaccines are administered as naked DNA or together with carriers, (including cationic lipids, liposomes, polymeric microparticles) [9], usually delivered by either intramuscular (i.m.) needle immunization or intradermal (i.d.) injection via a physical method known as “gene gun”, which is a needle free device that delivers gold beads coated with DNA vaccine plasmids into the epidermal layer of skin [10,11]. Numerous studies have demonstrated that the methods of DNA delivery affect the range of cell types to be transfected and the type of immune responses observed (Fig. 1). For instance, i.m. injection of plasmid DNA mainly leads to transfection of myocytes and to a lesser extent local antigen presenting cells (APCs) [12]. This route of immunization induces Th1-type immunoresponses, due to the presence in the backbone structure of plasmid DNA of unmethylated cytidine-phosphate-guanosine (CpG) motifs, that are recognized by Toll-like receptor (TLR)9 [13]. Ligation of TLR9 has a dual effect: (i) in macrophages, B cells and murine myeloid dendritic cells (mDC) lead to the activation of NF-κB, and the release of IL-12 and IFN-γ [14,15]; (ii) in plasmacytoid dendritic cells (pDC), TLR9 activation facilitates promotion of innate immunity and type I IFN production (i.e. IFN-β) [13]. On the other hand, i.d. gene gun-DNA immunization tends to transfect DC (Langerhans) directly [16], apparently bypassing TLR9 and inducing Th2dominated immune responses [7] (Fig. 1).

2.2. Advantages and potential concerns of DNA vaccines DNA immunization offers many advantages over traditional forms of vaccination: (i) in situ induction of native antigen expression; (ii) prolonged in vivo antigen production; (iii) efficient induction of humoral and cellular immune responses without the safety concerns of live attenuated vaccines; (iv) manufacturing in a relatively cost-effective manner; and (v) easier to produce, store and transport [17]. On the other hand, several issues have been raised regarding the

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Figure 1 Possible mechanisms of immune tolerance induction by DNA vaccines. Vaccines that aim to induce immune tolerance in MS consist of bacterial plasmids into which optimized gene sequences encoding one or several myelin antigens are incorporated. Plasmids are then delivered into small groups of cells either by intramuscular needle immunization (syringe) or by intradermal injection into the skin via gene gun. Afterwards, plasmids are taken up by different cell types, antigens processed and presented to the immune cells such that a specific immune response is generated or altered. In skeletal muscle, the main transfected cells are myocytes, however few antigen presenting cells (APCs) could also be transfected. When the transfected cells are myocytes, antigens are presented by two different mechanisms: (1) In a first pathway, antigen is processed and presented to T cells by myocytes; (2) In a second pathway, antigen produced by myocytes can by secreted and taken up by APCs, which then activate T cells. In the skin, the gene gun device delivers DNA directly into resident APCs (Langerhans cells and dermal cells). Mounting evidences from animal studies indicate that DNA vaccination induces different tolerance mechanisms depending on the route of immunization. Transfected myocytes take up the DNA and produce protein [1], but these cells usually lack the co-stimulatory molecules needed as part of the T cell activation process, thus leading to T cell anergy rather than full activation. Tolerance mechanisms associated with the second pathway are the loss of T cell costimulation via CD28, downregulation of IL-2 and IFN-γ production and reduced proliferative responses of T cells to myelin antigens [2]. Furthermore, intramuscular DNA vaccination induces IFN-β via activation of TLR9 by the CpG motifs present in the plasmid backbone. The induced IFN-β downregulates IL-12 expression (diminishing Th1 differentiation), IFN-γ and antigen-specific Th17 cell responses. Finally, intradermal vaccination using gene gun produces a protective effect by the induction of anti-inflammatory Th2 immune responses with the secretion of the regulatory cytokines IL-4, IL-10 and TGF-β. CpG: cytidine-phosphate-guanosine. TLR: Toll-like receptor. APC: antigen presenting cell.

safety of DNA vaccines, which include the potential to integrate into the genome of host cells, thus carrying a theoretical risk of insertional mutagenesis, the development of autoimmunity and the possibility of antibiotic resistance. Nevertheless, to date, DNA vaccines do not show significant levels of integration into host cellular DNA [18–20], and there is no convincing evidence of autoimmunity developing in association with their administration [21,22].

3. Tolerogenic vaccination The purpose of any vaccination is to produce protective humoral or cellular immune responses against target antigens that persist over a long period of time. These long-lasting immune responses

are mainly mediated by the induction of immunological memory, which improves immunity in the T and B lymphocyte subsets. Depending on the route as well as the dosage of administration, exposure to certain molecules will either stimulate effector responses or induce immune tolerance, a decision highly dependent on factors such as the signals that T cells receive from APCs and the surrounding environment existing during the activation process [23]. For example, signals through costimulatory receptors (CD28) and proinflammatory cytokines (IL-1 or IL-6) will favor inflammatory immune responses. In contrast, inhibitory receptor signals (cytotoxic Tlymphocyte-associated 4 or PD-1) or anti-inflammatory cytokines (IL-10 or transforming growth factor [TGF]-β) will favor immune tolerance [24].

DNA-based vaccines for multiple sclerosis: Current status and future directions Although DNA-based immunization research has largely focused on eliciting protective immunity against a variety of infectious pathogens, this approach may also be applied to other immune-related disorders such as autoimmune diseases. In this regard, DNA vaccines to treat autoimmune diseases should work as a “negative” form of vaccination, inducing tolerance rather than stimulation of immune responses to target antigens. DNA vaccines may induce immune tolerance by any number of potential mechanisms including anergy and deletion or induction of T cells to differentiate to a less inflammatory effector phenotype. In addition, regulatory T cells can actively suppress reactivity toward certain antigens (Fig. 1). The use of DNA vaccines to induce tolerance is relatively new compared with the use of peptides and proteins. Several reports have employed different neuroantigens such as MBP, PLP, and MOG, either the full-length sequences or individual encephalitogenic epitopes from these proteins. The use of whole proteins offers the potential to induce tolerance in T cells recognizing diverse epitopes within the same molecule [25]. Besides, as previously described by Fissolo et al. [26], DNA vaccines can encode different antigenic epitopes in the alternative reading frames of the same DNA sequence, delivering an extended spectrum of immunogenic information in a single DNA vaccine.

4. DNA vaccines in EAE There have been a number of studies using DNA vaccines in several autoimmune animal models [27]. Particularly, in EAE different strategies have shown some level of efficacy at inhibiting the disease, although in many cases this has only

Table 1

79

been amelioration rather than suppression of the disease, and for some cases DNA vaccines were associated with worsening of the clinical outcome [28–43]. Different molecules encoded by the DNA vaccines have been tested in EAE, including cytokines, chemokines, TCR peptides and myelin antigens. Studies in which a specific myelin autoantigen either alone or in combination with cytokines or immunomodulatory sequences were targeted by DNA vaccines are reviewed herein and summarized in Table 1.

4.1. Antigen-specific immune modulation Different studies have used autoantigens implicated in the pathogenesis of the disease, with the ultimate goal of inducing tolerization or downregulation of the antigenspecific autoreactive immune responses. Since EAE can be induced by different myelin proteins, such as MBP, PLP and MOG, different plasmid DNA vaccines expressing relevant sequences from each of the above mentioned pathogenic autoantigens have been tested. In 1997, Ramshaw et al. [28] achieved such a goal by administering MBP using a particlemediated gene transfer technology, delivering a plasmid DNA vaccine by gene gun into the skin. In this report, the authors demonstrated that gene gun DNA immunization preferentially induced Th2-type responses and prevented the development of EAE [28]. The efficacy of DNA vaccines in preventing EAE was further demonstrated when minigenes coding for encephalitogenic MBP peptides were shown to protect from subsequent EAE induced with the corresponding MBP peptide [29]. In this study [29], it was also observed that

Summary of the more relevant studies using plasmid DNA in EAE.

Transgene

Induced pathway of immune response

Disease outcome

Ref.

MBP MBP epitope PLP epitope PLP, PLP epitope MOG

Shift to a Th2 response Reduction of T cell reactivity against MBP Anergy induction, decreased IFNγ and IL-2 secretion Augmented PLP-specific lymphoproliferative responses Development of a cytopathic autoantibody response against MOG Enhanced type 1-like response, FasL downregulation Immunomodulatory effects exerted by induced IFNβ Downregulation of Ag-specific Th17 cell responses produced by the induced IFNβ Induction of peripheral tolerance, Th1 response Decreased/increased production of IFNγ IL-2 and proliferative responses Reduced IFNγ production Induction of a local T1 cytokine milieu by immunostimulatory (ISS) sequences protects from EAE Skewing of autoaggressive T and B cell responses toward a Th2 phenotype, decreased epitope spreading Shift cytokine profile to Th2 type Decreased epitope spreading of autoreactive B-cell responses Elevation of Ag-specific IL-10 producing regulatory T cells (tr1)

Improvement Improvement Improvement Worsening Worsening

[28] [29] [30] [31] [32]

Improvement Improvement Worsening

[33] [34] [35]

MOG epitope MOG epitope MOG epitope + siRNA-IFNβ PLP PLP, MOG MBP epitope-Fc of IgG MBP epitope-ISS, MBP-ISS + IL-4, IL-10, TNFα PLP, MBP, MOG, MOG + IL-4 + GpG-ODN MOG + IL-4, PLP + IL-4 PLP, MBP, MOG, MOG + IL-4 MBP + IL-10

Improvement/worsening [36] Improvement/worsening [37] Improvement Improvement

[38] [39]

Improvement

[40]

Improvement Improvement Improvement

[41] [42] [43]

Th1: T helper-1 cells; Th2: T helper-2 cells; Th17: T helper-17 cells; MBP: myelin basic protein; PLP: proteolipid protein; MOG: myelin oligodendrocyte protein; IL: interleukin; IFNβ: interferon-γ; IFN-γ: interferon-γ; Ag: antigen; ISS: immunostimulatory sequences; FasL: Fas ligand; Treg: regulatory T cells; EAE: experimental autoimmune encephalomyelitis; siRNA: short interfering RNA.

80 DNA vaccination with sequences for a specific myelin peptide did not cross-tolerize against other encephalitogenic MBP or MOG peptides. However, conflicting results have been observed in EAE studies using DNA vaccination with PLP and MOG as self-antigens. For instance, Ruiz et al. [30] demonstrated resistance to PLP 139–151-induced EAE in SJL/J mice that were prevaccinated with a DNA construct encoding the immunodominant PLP 139–151 determinant. Conversely, Tsunoda et al. [31] showed enhanced peptidespecific EAE induction in SJL/J mice prevaccinated with constructs encoding either the whole PLP protein or its encephalitogenic peptides. With regard to MOG-DNA vaccines, mice immunized with this gene developed an exacerbated form of EAE when challenged with either MOG or an unrelated encephalitogen [32]; however, a favorable clinical outcome was observed as a result of DNA vaccination with a MOG epitope (MOG91–108) [33,34]. The underlying mechanism for this protective MOGDNA vaccination included an induction of the antigenspecific anti-viral cytokine IFN-β and the downregulation of Ag-specific Th17 responses [34]. These observations were further substantiated by studies using short interfering RNA (siRNA) to IFN-β that completely abrogated the protective effects of the vaccine [35]. Some studies have shown significant differences in the kinetics of development of EAE tolerance in response to vaccination with different DNA-encoding myelin antigens. The outcome of the experiments (improvement versus worsening) was dependent upon the interval of time that elapsed between vaccination and subsequent sensitization with the encephalitogenic antigen. For example, early sensitization for EAE (4 weeks after DNA vaccination) with PLP139–151 caused recipient mice to develop a severe, exacerbated form of disease (in comparison to control mice), whereas late sensitization (N 10 weeks) resulted in a milder, ameliorated disease course [36,37]. On the contrary, DNA vaccination with a MOG-encoding construct prevented EAE for both early and late sensitization. Since PLP and MOG require different MHC presentation and induce different EAE models, these results point to potential differences in immune system requirements for efficient DNA-induced amelioration of the autoimmune response [37]. Several approaches have examined the effect of the addition into the plasmid of different DNA sequences in order to enhance the efficacy of DNA vaccines. For example, in a rat EAE model, Lobell et al. [38] showed inhibition of MBP-induced EAE and decreased production of the Th1 cytokine IFN-γ with a construct encoding the immunodominant MBP68–85 fused to an IgG binding domain. The authors hypothesized that binding of the hybrid gene product to IgG could target the antigen to Fc receptors and alter the presentation of MBP68–85 which results in a beneficial effect. In a later study, the same group reported that addition into the plasmid backbone of immunostimulatory sequences known as CpG, could activate the immune system acting through Toll-like receptors and produce a protective effect after vaccination with a MBP-DNA vaccine [39]. In contrast to this approach, Steinman et al. [25], by the elimination of these immunostimulatory CpG motifs and the addition of immunomodulatory GpG oligodeoxinucleotides enhanced the efficacy of various autoantigen-encoding DNA vaccines via reduction of non-specific proinflammatory responses [40].

N. Fissolo et al. Furthermore, delivery of myelin antigens together with anti-inflammatory cytokines, proved to enhance the suppressive effects of DNA vaccination on EAE severity, either by shifting the phenotype of autoreactive T cells to protective Th2 responses when IL-4 was used [40–42], or by suppressing EAE through the induction of Ag-specific IL-10-producing regulatory T cells (Tr1) following the addition of the IL-10 sequence into the plasmid DNA [43]. Given the differences in the model systems, constructs, and experimental designs, distinct mechanisms may account for the tolerance induction observed in each study.

5. Clinical trials of DNA vaccines in MS Thus far, only three clinical trials of DNA vaccines have been reported in autoimmune diseases, two of them in MS and one in type 1 diabetes. The first report in MS corresponds to a phase I/II randomized, double-blind, placebo-controlled trial with BHT-3009, a DNA vaccine encoding the full-length MBP molecule, driven by a human cytomegalovirus (CMV) promoter, and with an altered backbone plasmid [44]. Three different doses (0.5, 1.5 and 3 mg) of BHT-3009 or placebo were administered intramuscularly at weeks 1, 3, 5, and 9 to 30 MS patients (11 RRMS and 19 SPMS) who were not in any other disease modifying therapies [44]. BHT-3009 was found to be safe and well tolerated at all three doses tested, and all observed adverse events were considered to be mild to moderate in severity and brief in duration. The number of gadolinium-enhancing lesions and the gadolinium lesion volume were decreased in BHT-3009 treated patients compared with the placebo group, however, differences did not reach statistical significance. Interestingly, a decrease in the proliferation of IFN-γ-expressing CD4+ T cells specific for myelin epitopes from MBP, PLP and MOG, but not to control antigens was observed in patients during treatment with BHT-3009. In three patients with follow-up lumbar puncture, BHT-3009 vaccination was associated with a significant decrease in the cerebrospinal fluid autoantibody titters specific to MBP. Of note, BHT-3009 crosstolerized to other myelin antigens, as reflected by the reduction in the autoantibody titers observed for PLP. In summary, the DNA vaccine BHT-3009 was safe and effective in the induction of antigen-specific tolerance to myelin proteins in MS patients. However, treatment failed to show a significant clinical impact on lesion reduction at any dose tested. In a more recent randomized, placebo-controlled phase II trial, 289 RRMS patients who were not on any other disease modifying therapies were treated for a total of 44 weeks with BHT-3009 administered intramuscularly at two different doses, 0.5 mg and 1.5 mg, or placebo [45]. The DNA vaccine did not induced serious adverse events compared with the placebo group. There was a statistically significant reduction in the mean volume of enhancing lesions at week 48 in the group of patients treated with 0.5 mg BHT-3009 compared with placebo. Trends towards a reduction in the 4-week rate of occurrence of new gadolinium-enhancing lesion were also observed in 0.5 mg BHT-3009 when compared with placebo. However, no significant differences were observed between groups in clinical outcomes such as relative risk for relapse, annualized rate of relapse, or time to first relapse [45]. Of

DNA-based vaccines for multiple sclerosis: Current status and future directions note, the greatest reduction in the number of new gadolinium-enhancing lesions was observed in patients showing a high degree of anti-MBP reactivity in CSF samples obtained at baseline. In a subgroup of patients with followup CSF samples, significant reductions in the autoantibody titters to myelin autoantigens were observed in patients on 0.5 mg BHT-3009 compared with the placebo group. By contrast, treatment with 1.5 mg BHT-3009 was associated with an increase in titters to PLP peptide epitopes. This observation is in line with previous reports indicating that chronic low doses of an antigen are not immunogenic but might rather be tolerogenic, phenomenon known as “lowzone tolerance” [46]. Similar to the phase I/II trial, the decrease observed in the anti-myelin T-cell activity was not restricted to MBP-specific T cells and also included T cells reactive against other autoantigens such as PLP, MOG, or αβ crystalline. Patients enrolled in this phase II study had an offtreatment follow-up of year. Interestingly, brain lesion activity on MRI measured 18 months after randomization (6 months after cessation of therapy) showed a further decrease in disease activity with both doses of BHT-3009. These observations suggest that continued dosing of the DNA vaccine could further improve its efficacy [47].

6. Future directions for the use of DNA vaccines in MS To date, DNA vaccines tested for MS treatment have shown some level of efficacy, but still failed to show significant effects on lesion reduction or relapse rates [44,45]. Consequently, it is essential to improve the strategies for MS treatment based on DNA vaccines. Several technical improvements have contributed to increase the interest in the DNA vaccine field, including gene optimization strategies and more effective delivery approaches. At the same time, the development of vectors coding for relevant disease autoantigens is crucial for the efficiency of the DNA vaccine approach as a therapy for MS. Appropriate levels of antigen expression following DNA vaccination are critical to achieve efficient priming of an immune response [48] as well as tolerance induction [49]. There are several ways by which antigen expression can be improved for the DNA vaccine platform. One of the most efficient ways is through the use of codon optimization. A complete redesign of entire gene sequences could assure an effective transcription and translation of the antigenic protein and, consequently, maximize protein expression. Redesign strategies include modification at translation initiation regions, alteration of mRNA structural elements and use of different codon biases [50], all changes without modifying the amino acid sequence of the encoded protein. Recent reports demonstrate that codon optimization was shown to significantly increase the immune response to the DNA immunogens, reducing the amount of DNA necessary to induce the immune responses [51]. One of the potential advantages associated with DNA vaccination includes the possibility of modulating Th1 or Th2 responses via the alteration of vaccination protocols [52]. Studies in EAE and MS have shown that i.m. vaccination with

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plasmid containing myelin antigens is able to induce tolerance. Although literature on gene gun i.d. immunization is scarce, this delivery method has successfully been applied to treat EAE [28] and other autoimmune disease models such as type 1 diabetes in non-obese mice [53]. Besides, biodistribution studies showed that only a small fraction of the total DNA injected by i.m. injection is not degraded by enzymes in the extracellular fluids [54]. Consequently, different approaches have been developed to encapsulate and protect DNA, including the gene gun i.d. immunization by particle trapping and high-velocity delivery, which has the ultimate goal of introducing the plasmid directly into the cytosol of target cells, thus avoiding DNA degradation. Furthermore, muscle is not considered to be a viable site for antigen presentation, as it contains few, if any, dendritic cells, macrophages, and lymphocytes [55]. In addition, it has been well established that skin-associated lymphoid tissues harbor specialized cells which enhance immune responses, and that i.d. gene administration induced more profound immune responses than i.m. gene delivery [56]. Finally, immune responses observed following i.m. vaccination are of Th1 type whereas those produced after i.d. immunization are predominantly of Th2 type. Taking all this into account, gene gun i.d. vaccination with DNA encoding for multiple myelin antigens may result in more efficient strategies to switch on-going pathogenic immune responses into protective tolerogenic responses in MS patients. As mentioned before, emerging data from clinical trials with DNA vaccines encoding the encephalitogenic autoantigen MBP are promising and resulted in a significant reduction in disease activity in a subgroup of MS patients [44,45]. However, the array of autoimmune responses in MS challenges the idea that tolerizing therapies targeting only a few epitopes or even the whole protein will show efficacy. Therefore, DNA vaccination with constructs encoding multiple myelin antigens and/or novel genes identified as potential therapeutic targets may prove to be more efficient to treat MS than DNA-encoding single proteins, thus avoiding epitope spreading, a phenomenon which is believed to perpetuate autoimmune responses [42]. In this regard, DNA vaccination may result in a promising therapy to eliminate immune responses directed against a wide array of antigens in MS.

7. Conclusions This review highlights the potential for DNA vaccination to induce immune tolerance in MS. Although MS is considered to be an immune-mediated disease, currently used immune therapies have shown a lack of efficacy to inhibit disease progression. Thus, there is a need for better therapeutic strategies, such as antigen-specific therapies that selectively target the pathological lymphocytes involved in the disease. A significant volume of data from the animal model reinforces the protective effect of DNA vaccines in EAE. Furthermore, two recent clinical trials with DNA vaccines in MS have shown promising results at reducing MRI-measured disease activity in patients with relapsing-onset MS. Although results obtained thus far are encouraging, the hypothesized mechanisms of action of many DNA vaccines have been

82 studied and elucidated primarily in EAE. Thus, future strategies combining both clinical studies and basic research should aim to elucidate the mechanisms of action of DNA vaccines in MS, in order to provide a greater understanding of the disease that could be exploited for further development and improvement of immune therapies in MS and other autoimmune diseases.

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