recent advances in dna vaccines for autoimmune diseases

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The immune system protects the body from potentially harmful substances by recognizing and responding to antigens. Classically, it is believed that discrimination between self and nonself constituents is fundamental for trigger-ing an effective immune response that is selective for exogenous agents that can damage the body. In this context, autoimmunity is understood as a failure of the immune system to recognize its own components as self, which results in an aberrant and harmful immune response towards its own cells and tissues.

In recent decades, a great deal of information has accumulated regarding immunological patho-genesis, thereby enabling a clearer understanding of the mechanisms involved in organ-specific and systemic autoimmune diseases. Concomitantly, we witnessed enormous progress in immunology that resulted in new strategies and tools for the modulation of the immune system, enabling the investigation of newer and ingenious protocols to control autoimmunity.

Immunological alterations found in autoimmunityThe etiology and pathogenesis of autoimmune diseases has long been a mysterious subject involving complex genetic and environmental

interactions. A recent review and pooled ana lysis of the past 30 years of research on the role of MHC in six genetically complex diseases (mul-tiple sclerosis [MS], Type 1 diabetes [T1D], systemic lupus erythematosus, ulcerative colitis, Crohn’s disease and rheumatoid arthritis [RA]) supported established MHC disease associations and also identified new predisposing variants [1]. Expression of other genes located out of the MHC complex, such as CTLA-4, Foxp3, LYP and TNF, has also been linked to autoimmunity. The environment can also affect autoimmunity in advantageous or deleterious manners. Some autoimmune diseases appear after infection by pathogens whose proteins present cross-reactivity with host epitopes [2]. In addition, by causing tis-sue damage, pathogens can liberate and expose host proteins that are usually sequestered, break-ing their self-tolerance [3]. Conversely, accumu-lating evidence has demonstrated that certain infections can prevent the development of auto-immune diseases. This possibility, termed the hygiene hypothesis, was initially postulated to explain the inverse correlation between the incidence of infections and the rise of allergic diseases, particularly in the developed world [4]. Recently, this hypothesis has been extended to encompass protection against autoimmunity. Of

Celio L Silva†, Vânia LD Bonato, Rubens R dos Santos-Júnior, Carlos R Zárate-Bladés and Alexandrina Sartori†Author for correspondenceAvenue Bandeirantes 3900, Depto de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, CEP 14049–900, Ribeirão Preto, São Paulo, Brazil Tel.: +55 163 602 3086 Fax: +55 163 602 3238 [email protected]

Vaccination is one of the most powerful health tools available owing to its ability to confer protection against various diseases. The long-term impact of such protection in terms of public-health savings is nearly incalculable and becomes even more evident when considering if the vaccination concept is extended to the therapeutic potential of a given molecule. In this sense, DNA vaccines are especially important tools with enormous potential owing to the molecular precision that they offer. The properties of the plasmid DNA molecule in terms of stability, cost–effectiveness and lack of cold-chain requirement are additional advantages over traditional vaccines and therapeutics. We focus on the current knowledge of autoimmune mechanisms, engineering of DNA vaccines and attempts that have already been made in order to intervene in autoimmune processes. Our experience with a genetic vaccine containing the heat-shock protein gene (hsp65) from mycobacteria is also described.

Keywords: autoimmunity • DNA vaccine • tolerance • Treg cell

Recent advances in DNA vaccines for autoimmune diseasesExpert Rev. Vaccines 8(2), 239–252 (2009)

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Review Silva, Bonato, Santos-Júnior, Zárate-Bladés & Sartori

various candidate agents, particular emphasis has been placed on the protective effects of worms [5] and mycobacteria [6]. The mechanism by which these organisms prime immunoregulation is currently being unraveled. It has been suggested that these organisms can stimulate a specific pattern of dendritic cell (DC) maturation that enables them to trigger differentiation of Treg cells rather than that of Th1 or Th2 effector cells [7].

A complete description of immunopathological findings is beyond the scope of this review. However, we will briefly high-light the most relevant characteristics of arthritis, diabetes and MS, which have been the targets of our research.

Rheumatoid arthritis is a chronic inflammatory disease that manifests clinically as a symmetric polyarthritis associated with swelling and pain in multiple joints, and is characterized by syn-ovial hyperplasia and progressive joint destruction [8]. Among the numerous cell types present within the inflamed joint, there is a massive influx of T cells (Th1 cells have been classically described), B cells, fibroblast-like synoviocytes, macrophages and DCs [9]. A stronger contribution of Th17 cells was described recently [10]. Experimental evidence from arthritis animal mod-els clearly indicates that Treg cells contribute to the control of disease severity [11]. Enrichment of CD4+CD25+ T cells in the synovial fluid of patients with RA has been described [12] and, interestingly, these cells suppressed proliferation and cytokine production [13].

Type 1 diabetes is a chronic autoimmune disease associated with the generation and activation of T cells reactive against pancreatic B-cell autoantigens. Self-reactive CD4+ and CD8+ T lymphocytes infiltrate the pancreas, causing increasingly severe insulitis that ultimately destroys insulin-producing β-cells present in islets. Islet destruction is characterized as a silent but progressive proc-ess that may remain undetected for a prolonged period. Clinical symptoms usually appear later, when almost 80% of β-cells have already been destroyed [14].

There are several lines of evidence supporting the contribution of Treg cells in the pathogenesis of T1D. Interestingly, adoptive transfer of Treg cells can be employed not only to prevent diabetes, but also as a therapy for established experimental diabetes [15]. Much of the knowledge acquired in this field has been obtained in the nonobese diabetic (NOD) mouse model [16]. Deficiency in NOD antigen-presenting cell (APC) function has been blamed for diabetes development in these animals [17]. On the other hand, the human situation appears to be much more complex. For example, natural Treg cells are not under-represented in T1D, and Treg function is likely to be suboptimal in a subset of patients [18]. A different perspective was recently raised by the discovery that self-reactive cells can be resistant to regulation by Treg cells [19].

Multiple sclerosis is the most common inflammatory demyeli-nating and neuro degenerative disorder of the CNS in humans. Substantial evidence supports the involvement of both innate and specific immunity in the response against several self-antigens associated with myelin and oligodendrocytes [20]. The hallmark of this pathology is the infiltration of different cellular types including Th1 and Th17 T-cell subsets and B lymphocytes into

the brain and spinal cord [21]. Involvement of Treg cells has been demonstrated lately, mainly in investigations of experimental autoimmune encephalo myelitis (EAE), which is a largely accepted animal model of MS. For example, deletion of Treg cells facili-tated spontaneous autoimmune encephalitis in mice, whereas aug-mentation of Treg function alleviated or even prevented disease development. Even though the number of CD4+CD25+ Treg cells does not differ between patients with MS and healthy controls, these cells are functionally impaired in patients [22].

Strategies of the immune system for avoiding autoimmunityT- and B-cell repertoires are generated in a quasirandom process of gene rearrangement that initially occurs in the absence of foreign or self-antigens. As a consequence, in addition to its ability to recognize an enormous variety of foreign epitopes, the immune system is also able to recognize self components. A range of dif-ferent mechanisms involving B and T cells in both central and peripheral lymphoid organs is necessary to insure self-tolerance. Failure of these mechanisms can result in autoimmune diseases. Even though B cells have recently been linked to autoimmunity, the contribution of T-cell dysregulation has been investigated far more thoroughly [23]. We will therefore briefly describe the primary mechanisms responsible for self-tolerance in this cell population.

The thymus contributes to self-tolerance in at least three dis-tinct ways: negative selection, receptor editing and generation of Treg cells. Negative selection is the clonal deletion by pro-grammed cell death of CD4+ and CD8+ T cells that bind to self-epitopes presented by MHC molecules in the thymus. This unique self-representation in the thymus occurs mainly in thymic epithelial cells and was only recognized recently. It is regulated by a gene called the autoimmune regulator and has been referred to as promiscuous gene expression [24,25].

Another self-tolerating mechanism present in the thymus is active genetic correction of self-reactive receptors by second-ary DNA recombination. Briefly, in this process, also known as receptor editing, signaling through an autoreactive antigen receptor promotes further receptor–gene recombination; in this way self-reactive receptors are destroyed and substituted with harmless ones, thereby eliminating cellular autoreactivity without eliminating the cell itself [26]. These two mechanisms are not, however, sufficient for silencing all self-reactive T-cell clones; some of these cells are still found in healthy donors. It is believed that these self-reactive T cells are either anergized or counter-regulated in the peripheral lymphoid organs of healthy individuals by Treg cells [27]. Two families of Tregs have been identified and characterized as naturally occurring or adapta-tive. Both originate in the thymus but the first subset achieves complete functional maturation in the thymus itself, whereas the second functionally matures in the periphery [28]. Recent attention has focused mainly on naturally occurring CD4+ Treg cells expressing the α-chain of the IL-2 receptor (CD25+). In most instances, these CD4+ CD25+ Treg cells also express GITR, cytotoxic T lymphocyte antigen A (CTLA)-4 and L-selectin (CD62L) [29–31]. The Foxp3 gene, a member of the forkhead/

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ReviewRecent advances in DNA vaccines for autoimmune diseases

winged-helix family of transcriptional factors, has been described as fundamental in controlling the development and expression of suppressive phenotypes in Treg cells [32–34]. Very recently, however, the necessity of Foxp3 expression in human T cells for suppression has been debated [35].

A fundamental aspect still waiting to be disclosed is the immuno-logical scenario in which the induction of Treg cells prevails. Recent data indicate that distinct subsets of DCs exist in the intestine and spleen and they are specialized to induce differentiation of Foxp3+ Treg cells in those microenvironments [36,37].

Mechanistic studies suggest that TGF-β appears to be essential for regulating peripheral Treg number and also their immuno-suppressive effects in vivo and in vitro [38]. Also in this context, a crosstalk between DCs and Treg cells would explain a very interest-ing idea called the hygiene hypothesis. This hypothesis suggests that increases in chronic inflammatory disorders (allergies, inflamma-tory bowel diseases and autoimmunity) in developing countries are partially attributable to decreased contact with certain organisms. These environmental saprophytes, such as helminths and myco-bacterias, would have the ability to induce Treg cells by directly interacting with pattern recognition receptors on DCs [7].

The mechanisms of Treg action remain poorly understood and contentious. As reviewed recently, suppressive routes can be divided into three categories: cell–cell contact, local secretion of inhibitory cytokines and local competition for growth factors [39]. Surface molecules, such as membrane-bound TGF-β, cytosolic molecules (Fas and granzyme B), lymphocyte activation gene 3 and CTLA-4 have being implicated in suppressive activity [40]. The modulation of cyclic adenosine monophosphate levels in tar-get cells, which selectively inhibits the production of cytokines, such as IL-2 and IFN-γ, is an inhibitory pathway of interest [41]. TGF-β and IL-10 are frequently associated with effector func-tions of Treg cells [42,43]. Constitutive expression of CD25, in contrast to induced expression in naive T cells, enables Treg cells to consume IL-2 and thus trigger cytokine deprivation-induced apoptosis [44]. The growing number of inhibitory mechanisms linked to Treg activity suggests a multidirectional strategy for immune regulation. Sojka et al. suggested that each inhibitory mechanism is related to, and modulated by, the inflammatory milieu and the extent of the immune response [40].

The presence of defective Tregs in many autoimmune dis-eases including MS, T1D, psoriasis, myasthenia gravis and RA is consistent with a relevant role for these cells in the control of self-reactivity and may open new avenues for therapy.

Experimental approaches to avoid or treat autoimmunityTo date, most approved therapies for treatment of autoimmune diseases are based on global inhibition of immune inflammatory activity. Although this approach is partially effective, numerous side effects are incompatible with long-term patient survival. One of the goals of ongoing research is to avoid undesirable general suppression by determining specific self-tolerance. Four proto-cols are currently employed to induce protein–peptide-specific immune tolerance: soluble peptide-induced tolerance, DNA

vaccination-induced tolerance, coupled cell-induced tolerance and altered peptide-induced tolerance. A very complete review, including experimental aspects and clinical trials, was recently published by Miller et al. [45]. We will focus on new strategies to employ DNA constructs for the treatment of autoimmunity.

DNA vaccinesEdward Jenner provided the initial and fundamental step towards the development of the first vaccine against small-pox in the 18th century. Almost a century later, Louis Pasteur presented experimental evidence, with tremendous impact on the future field of vaccinology [46–48]. Another century after Pasteur’s experiments, Wolff and colleagues demonstrated that the administration of naked DNA into mouse skeletal muscle resulted in significant expression of reporter genes in muscle cells [49]. Soon after, in 1993, Ulmer and colleagues demon-strated that the intra muscular injection of plasmid DNA encod-ing an influenza viral protein induced antigen-specific cytotoxic T lymphocyte (CTL)-mediated protection against subsequent challenge with live influenza virus [50]. These studies demon-strated for the first time that vaccines not constituted by whole microorganisms were able to stimulate CTL-mediated immune responses. Since then, the prophylactic potential of DNA vac-cines has been evaluated in a variety of experimental models of infectious diseases [51]. Our group described, for the first time, that a DNA vaccine initially employed to prevent a disease [52], could also be used to treat it [53], thus opening a new frontier for the application of genetic vaccines (Figure 1). These vaccines are probably a unique health resource that can be engineered and applied to a broad array of diseases, including infections, tumors, autoimmune pathologies and other disorders, as has been demonstrated by different laboratories [54,55]. The proper-ties of the plasmid DNA molecule in terms of stability, cost–effectiveness and nonrequirement of cold-chain represent addi-tional advantages over traditional vaccines and therapeutics. DNA vaccines consist of a foreign gene of interest (antigen or an immunomudulator molecule) cloned into a bacterial plasmid. Some elements are essential in recombinant plasmid construc-tion for DNA vaccines, including a strong eukaryotic promoter for expression of gene products by mammalian host cells, an Escherichia coli origin of replication that enables the production of large plasmid copy numbers in bacteria, a bacterial antibiotic resistance gene for plasmid selection during bacterial culture, polyadenylation sequences for stabilization of mRNA transcripts and CpG sequences that confer immunostimulatory properties to DNA constructs [54].

Plasmid DNA captureThe mechanism of plasmid DNA capture is not well understood. It had been reported that plasmid DNA can be taken up by macropinocytosis or by DNA-binding cell-surface proteins, ezrin and moesin [56] and class A scavenger receptor MARCO [57]. Recently, Trombone and colleagues observed that clath-rin-mediated endocytosis is involved in DNA plasmid capture by mouse macrophages [58]. Once internalized, plasmid DNA


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must exit endosomal–lysosomal compartments. DNA fragments larger than 1 kb appear to remain in the cytoplasm, in contrast to smaller DNA frag-ments (250 bp), which are able to diffuse into the nucleus [59]. It is likely that endosomal–lysosomal degradation and cytoplasmic barriers constitute the main factors preventing the transport of intact plasmids into the nucleus. Nuclear translocation of DNA requires either the disassembly of the nuclear envelope or active nuclear transport via the nuclear pore complex [60]. Transfection efficacy is one of the most significant obstacles found in plasmid DNA delivery. In this context, optimization of vectors or tools that will enable efficient uptake of DNA delivery is necessary for the improvement of DNA vaccine efficacy [61].

Activation of innate & specific immunity by DNA vaccinationCpG sequences in the plasmid backbone play an adjuvant role in DNA vaccines. These sequences represent pairs of unmethylated CpG dinucleotides on the same DNA strand. They must present flank-ing regions of two purines at the 5́ end and two pyrimidines at the 3´ end. CpG sequences are char-acterized as a pathogen-associated molecular pattern and are recognized by Toll-like receptor (TLR)9, a pattern recognition receptor [62]. TLR9–CpG inter-action occurs in the endosome [63] and leads to the activation of several transcription factors, result-ing in cytokine and chemokine expression [64–67]. CpG–oligodeoxynucleotides (ODNs) induce secre-tion of proinflammatory cytokines from monocytes, macrophages and DCs [63,64]. They also activate natural killer cells [68] and B cells [69] and confer to DNA plasmids the role of a natural Th1 adjuvant, as they stimulate IL-12 secretion.

Distinct CpG–ODNs have been engineered; either the number or the position of CpG di nucleotides appears to largely influence the extent of cellular activation [70]. Recently, our group dem-onstrated that recombinant plasmid DNA used as a vaccine against experimental TB stimulated the secretion of IL-12 by human monocyte-derived DCs, as well as that of TNF-α, IL-6 and IL-10 by monocyte-derived macrophages. Despite the expression of TLR9 by both cell types, differences in DNA uptake were observed. The immuno-stimulatory potential of recombinant plasmid DNA was equivalent to that associated with the empty vector. However, only recombinant plasmid DNA was able to induce proliferation of peripheral blood mononuclear cells [71]. Recently, another DNA sensor present inside the cells and called TANK-binding kinase 1 was described. This molecule is Fi

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Review

DNA vaccineencoding tolerogenicprotein

Induction of specific tolerance Induction of Tregs

Induction of anti-inflammatory state

DNA vaccineencoding IL-4, IL-10,TGF-β or CTLA-4

DNA vaccinerich in CpGinhibitory motifs

DNA vaccine encoding Treg-inducer proteins:specific self-proteinsheat-shock proteins

Recent advances in DNA vaccines for autoimmune diseases

a non canonical IκB kinase that appears to play a key role in the immunogenicity of plasmid DNA [72].

At least three mechanisms seem to elicit T-cell responses during genetic vaccination:

Direct priming by somatic cells (myocytes and keratino-•cytes);

Direct transfection of somatic APCs, such as DCs;•

Cross-priming, in which recombinant plasmids transfect somatic •cells and/or APCs, and the secreted protein is then taken up by other professional APCs and presented to T lymphocytes.

The pathway employed for plasmid DNA delivery determines the pattern of the induced immune response. DNA adminis-tration by intramuscular or subcutaneous routes elicits cellu-lar immune responses, characterized by IFN-γ production and increased levels of IgG

2a. By contrast, DNA administration by

the biolistic gene gun approach mainly evokes humoral immune responses, characterized by IL-4 secretion and high levels of anti-gen-specific IgG

1 [73]. Improved efficacy of DNA formulations

have been obtained by the use of different administration routes, DNA plasmid encapsulation and, more recently, the prime–boost strategy.

Tolerance inductionAn array of strategies based on different mechanisms are being tested to prevent or treat autoimmune diseases. Anergy induction and change from a Th1 to a Th2 profile are classically described as mechanisms that underlie tolerance induction [74]. Stimulation of distinct types of regulatory cells, such as, for example, antiergotypic T cells [75], natu-ral killer T cells [76], Tr1 and Foxp3 Treg cells and also Th17 regulatory cells [77,78], are being thoroughly investigated and hold much hope for the future control of autoimmune diseases.

Similar to classical immunization proce-dures, DNA vaccines can also be used for both the induction of an effective immune response as well as tolerance elicitation. In the case of genetic constructions, this abil-ity to induce tolerance derives mainly from the chosen vaccination route, the versatil-ity of the construction itself and from the employed immunization protocol. DNA delivery by mucosal or intravenous routes has been proven to be useful to control autoimmunity diseases [79,80]. Soon after the discovery of DNA, scientists realized that these constructs were extremely versa-tile. They could, for example, be designed to carry any possible gene, including the

ones codifying for immunomodulatory molecules, and even for the association of various gene fragments. This understanding, associated with the discovery that the plasmid backbone content could also be modified, significantly enhanced their potential to be used for both immunogenicity or tolerogenicity purposes. Tolerance has been preferentially obtained by inserting genes coding for IL-4, IL-10, TGF-β and CTLA-4. Alternatively, the content of the plasmid vector can also be altered to reduce the number of immunostimulatory CpG motifs or to increase the number of immunoinhibitory CpG sequences (Figure 2). A plethora of questions, including the best sites for vaccination and the need for boosting procedures, is waiting for answers concerning the use of DNA constructs in the control of autoimmunity.

This versatility of DNA constructions has been elegantly explored to induce tolerance in experimental models of arthri-tis, diabetes and MS. Examples of these promising construc-tions to control autoimmune diseases can be observed in Table 1 and some of them are discussed in more detail in the text. Mice receiving a tolerance-inducing DNA-encoding type II collagen demonstrated reduced production of the proinflam-matory cytokines IFN-γ and TNF-α, suggesting the induction

Figure 2. Scientific rationale that supports the use of prophylactic or therapeutic DNA vaccines for controlling autoimmune diseases. DNA vaccines can be designed to include elements to trigger at least three different effects against autoimmunity: (1) induction of antigen-specific tolerance, (2) induction of Treg cells and (3) anti-inflammatory state. CTLA: Cytotoxic T lymphocyte antigen.



of anergy. Microarray ana lysis also revealed reduced spreading of auto antibody response [81]. Intramuscular delivery of plas-mid DNA encoding β-cell antigen glutamic acid decarboxy-lase, insulin or Hsp60 prevented insulitis and diabetes onset in NOD mice [82–85]. However, it is important to stress that some protocols do not protect against, and can even accelerate, T1D progression [86]. It is therefore critical to choose the correct target antigen and administration technique in order to pro-duce the required tolerance. Optimization of tolerance induc-tion by DNA vaccines has been better demonstrated in EAE. DNA encoding a single encephalitogenic epitope prevented the initial onset of EAE, and this protective effect was associated

with anergy in T auto reactive cells [87]. DNA vaccines encoding either proteolipid protein or myelin oligodendrocyte glycopro-tein in combination with IL-4 DNA was demonstrated to be an efficient strategy for shifting autoimmune encephalitogenic Th1 cells to the Th2 phenotype [88]. The use of many self-antigens (myelin basic protein [MBP], myelin oligodendrocyte glycoprotein and proteolipid protein) in a DNA cocktail, with or without a plasmid encoding IL-4, demonstrated promising results even when administered after hind-limb paralysis. This construction reduced both the spread of autoantibody responses to many epitopes and the relapse rate [89]. Bar-Or et al. con-ducted the first clinical trial to evaluate a DNA vaccine for the

Table 1. DNA vaccines tested in animal models of autoimmune diseases and clinical trials*.

Disease Model DNA vaccine-encoding genes Effect‡ Ref.

Diabetes Mice Mycobacterium leprae heat-shock protein 65 Protection [125]

Diabetes Mice BAX protein and glutamic acid decarboxylase Immunomodulation [134]

Diabetes Mice Membrane bound pre-proinsulin and mutant B7.1/CD40L

Immunomodulation [135]

Diabetes Mice Glutamic acid decarboxylase Prevention [136]

Diabetes Mice BAX protein and glutamic acid decarboxylase Prevention [97]

Diabetes Mice B7.1 mutant (CTLA-4 ligand) and pre-proinsulin Protection [137]

Diabetes Mice Human heat-shock protein 60 Inhibition [84]

Arthritis Mice Macrophage migration inhibitory factor and T-cell epitopes of tetanus toxoid

Protection [138]

Arthritis Mice M. leprae heat-shock protein 65 Immunomodulation [121]

Arthritis Rats CD25 Protection [75]

Arthritis Rats Human heat-shock protein 60 and M. leprae heat-shock protein 65

Inhibition [139]

Arthritis Mice Fas ligand Immunomodulation [140]

Arthritis Mice Type II collagen Therapy [81]

Arthritis Phase I trial DNA hepatitis B (ENGERIX) Safety and efficacy [141]

Systemic lupus erythematosus Phase I trial DNA hepatitis B (ENGERIX) Safety and efficacy [142]

Autoimmune uveitis Mice Interphotoreceptor retinoid-binding protein Protection [91]

Antiphospholipid syndrome Mice TNF-α Protection [142]

Experimental autoimmune encephalitis

Rats TNF-α Resistance [143]


Rats IL-4 plus suppressive CpG–ODN Suppression [144]


Rats IL-4 and self-peptide proteolipid protein 139–151 Protection [88]


Rats Myelin basic protein-peptide 68–85 and three ISS 5’-AACGTT-3’

Immunomodulation [145]

Multiple sclerosis Phase I and II trials

Human myelin basic protein (BHT-3900) Therapy [146,147]

Multiple sclerosis Phase I/II trial Human myelin basic protein (BHT-3900) Therapy [90]

*Shows some of the main reports of DNA vaccination in autoimmunity. ‡In the case of Phase I clinical trials, the objective of the studies were safety and efficacy, as is indicated in the Effect column of the table. CTLA-4: Cytotoxic T lymphocyte antigen 4; ODN: Oligodeoxynucleotide.



treatment of an autoimmune disease [90]. In that study, a DNA vaccine called BHT-3009 was engineered that encoded full-length human MBP. This vaccine was used to treat 30 patients with relapsing–remitting or secondary progressive MS. In addi-tion to being safe and well tolerated, the vaccine downregulated the specific immune response and also had a beneficial clinical effect as determined by MRI. The down regulation associated with this protocol was explained not only by the choice of MBP, which was a major autoantigen involved in MS, but also by modifications of the CpG content of the plasmid vector. In this case, the plasmid backbone was modified by reducing the number of immunostimulatory CpG motifs and by increasing the number of immunoinhibitory ones.

It is possible that tolerance-inducing DNA vaccines elicit Treg activation, as demonstrated by Silver et al. [91]. However, this was not systematically investigated. That study found that intravenous vaccination with a naked DNA vaccine expressing the uveitogenic retinal antigen interphotoreceptor retinoid-binding protein (IRBP) protected animals from developing uveitis after immu-nization with IRBP. Mechanistic studies revealed that the hypore-sponsiveness to IRBP was due to antigen-specific CD4+CD25+ T cells that expressed Foxp3, CTLA-4 and GITR.

Treg cell inductionThere has been a recent rebirth of suppressor cells as a central mechanism of immune regulation. These cells have been desig-nated regulatory T cells, and their possible use for restoring self-tolerance lost in autoimmune diseases has received a great deal of attention. A growing number of reports have demonstrated reduced functional activity of Tregs in many autoimmune dis-eases (MS, type II polyglandular syndrome, active RA, T1D, pso-riasis and myasthenia gravis) compared with cells from healthy donors. This subject was reviewed recently by Cools et al. [92]. Increased Treg activity is, thus, a logical therapeutic strategy for autoimmunity. Induction of Treg activity by DNA vaccination has been achieved using specific self-antigens and heat-shock proteins (HSPs) (Figure 2).

Induction by specific antigensMany procedures have been used to induce Treg activation by specific antigens. For example, the coinoculation of DNA and pro-tein vaccines, a protocol that is similar but distinct from prime–boost strategies, has resulted in immuno suppression character-ized by reduced antigen-specific T-cell-mediated responses [93,94]. Coadministration of two plasmids, one encoding MBP and the other IL-10, rapidly suppressed ongoing EAE. Tolerance included increased numbers of antigen-specific T cells produc-ing IL-10, as well as increased apoptosis of cells near high endo-thelial venules in the CNS. This effect was mainly attributed to Tr1 cells [95]. Silver et al. explored the concepts of privileged self-antigen and induced immunosuppression using an intravenous route [91]. Specifically, hydrodynamic intravenous injection of naked DNA was used to express IRBP in the periphery, result-ing in strong protection of vaccinated mice from uveitis. Based on the role of CTLA-4 in the induction and maintenance of

peripheral T-cell tolerance, Eggena et al. elegantly demonstrated that CTLA-4 and Treg cells act co operatively to maintain tol-erance in a transgenic diabetes mouse model, indicating that the function of CTLA-4 is independent of regulatory cells and that deficiency of both is mandatory for induction of autoim-munity [96]. Additional strategies for inducing protective self-tolerance have utilized the codelivery of proapoptotic molecules together with self-antigens [97], as well as more classic mucosal immunization [98].

Induction by HSPsOriginally, HSPs were identified as a group of proteins whose expression was induced by heat; however, other stimuli, includ-ing growth factors, inflammation and infection, are also able to induce their secretion [99]. HSPs are grouped according to their approximate molecular weight as small HSPs, Hsp40, Hsp60, Hsp70, Hsp90 and large HSPs. These proteins are expressed by prokaryotes, eukaryotes and plants, where they are found in vari-ous cell compartments including the cytosol, nucleus, endoplasmic reticulum, mitochondria and chloroplasts [100].

Selected HSPs, also known as chaperones, play crucial roles in the folding and unfolding of proteins, the assembly of multi-protein complexes, the transport and sorting of proteins into the correct subcellular compartments, cell-cycle control and signaling. These proteins are also important for the protection of cells against stress and apoptosis. Moreover, recently, HSPs have been implicated in antigen presentation, as they are thought to guide and transfer antigenic peptides to class I or II MHC molecules [101].

The observations of high-sequence homology between eukaryo-tic and prokaryotic HSPs and high immunogenicity of micro-bial proteins have prompted the hypothesis that HSPs could, via molecular mimicry, act as potentially dangerous autoantigens. Levels of HSPs and anti-HSP antibodies have been indicated in autoimmune inflammatory responses in diseases such as arthritis, MS and diabetes. However, it has also been demonstrated that mycobacterial Hsp60 actually protects against arthritis induced by Mycobacterium tuberculosis immunization [102]. Interestingly, this protective effect was also observed in other chronic inflam-matory disease models, including EAE, collagen-induced arthritis and diabetes [103–107]. A thorough investigation on this suppressive ability of mycobacterial Hsp60 revealed that only self-HSP cross-reactive peptides induced protection against adjuvant arthritis via activation of self-HSP reactive Treg cells [102]. Even though a detailed comparison between mammalian and mycobacterial Hsp is still missing, mammalian Hsp60 DNA is reported to be more effective than mycobacterial Hsp65 DNA [84]. Further evaluation proved that this anti-inflammatory activity of self-Hsp60 was a dominant function, rather than a possible deleterious effect. New protocols based on the usage of this protein (entire and fragments) or in the transfection of the encoding DNA were devised to induce activation and expansion of Treg cells. Peptides from HSPs, as has already been clearly demonstrated in arthritis and diabetes, demonstrated an outstanding potential to control autoimmunity and are already included in clinical trials [108–110].



In order to stress the complexity of this subject, we would like to highlight some particularities of this self-centered immunoregulatory circuit. For example, other HSPs, such as Hsp70 and Hsp90, presented similar immunoregulatory func-tion [110,111]. These different HSPs appear to work in a concerted effort to achieve higher levels of immunoregulation [110]. The possible existence of an immunoregulatory circuit triggered by DNA itself was not formally investigated. However, pathogen DNA or even the plasmid DNA used in the genetic constructions could, theoretically, start this circuit via their CpG content. This is supported by the demonstration of a direct binding between CpG–ODNs and commercially available human Hsp [112].

Our experience with DNAhsp65 in autoimmunity We initially described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65-kDa HSP (DNAhsp65) in experimental murine TB [52,53]. In addition, vari-ous HSPs, including M. leprae Hsp65, have been associated with immunomodulatory effects via priming of immune responses in different tumor models [113,114].

Our experiments with DNAhsp65 for autoimmunity were ini-tially conducted to confirm that this proposed TB vaccine would not trigger undesirable autoimmune side effects. This possibility existed due to the high homology of the mycobacterial Hsp65 encoded in the DNA vaccine with the corresponding mamma-lian Hsp60 [115–117], as well as the presence of CpG motifs in the plasmid vector, which together could trigger or exacerbate an autoimmune response [118–120]. Given the relevance of arthritis, diabetes and MS, and also the involvement of the anti-Hsp response in all three conditions, we investigated the effect of the DNAhsp65 vaccine in the corresponding experimental models. The potential of DNAhsp65 vaccination to induce or modulate arthritis was tested in mice genetically selected for either maxi-mal acute inflammatory reactions (AIRmax) or minimal acute inflammatory reactions (AIRmin) [121]. Mice immunized with DNAhsp65 or injected with the corresponding DNA vector (DNAv) did not develop arthritis, whereas pristane injection caused arthritis in 62% of AIRmax mice. These data were con-sistent with the findings of Vigar et al., who demonstrated that pristane injection resulted in a higher incidence of arthritis in AIRmax mice compared with AIRmin mice [122]. Interestingly, the incidence of arthritis in AIRmax mice injected with pristane was decreased by concomitant administration of DNAhsp65. Even more striking was the protective effect against arthritis observed when DNAhsp65 was administered after pristane in AIRmax animals. Most of this immunomodulatory effect was considered specific for Hsp65 because by day 150, the incidence of arthritis in DNAhsp65-vaccinated mice reached zero, in con-trast to the group injected with DNAv, which still displayed sig-nificant disease incidence. Histological analyses demonstrated no inflammatory infiltrates in DNAhsp65-vaccinated AIRmax animals [121]. A very clear downregulatory effect was triggered by both vaccine and vector against IL-12 production by splenic cells. Interestingly, this was associated with a concomitant rise in IL-10 levels that was more pronounced in animals injected

with the vaccine compared with those injected with the vector. Despite the lower incidence of arthritis generated by the Hsp65-specific immune response, the nonspecific role of DNA itself was also considered important. The immunostimulatory effects of DNA are more prominent than inhibitory effects and have been attributed to the CpG motifs present in its backbone, which stimulate the secretion of IL-12 and IL-6 by monocytes and DCs [123,124]. However, an inhibitory effect of CpG motifs has previously been described in an experimental model of diabetes and is associated with a decreased proliferative T-cell response to Hsp60 and its p277 peptide [85].

The potential of DNAhsp65 vaccination to induce or modulate diabetes was tested in NOD mice, which spontaneously develop insulin-dependent diabetes mellitus (IDDM) as a consequence of autoimmune destruction of the insulin-producing β-cells of the pancreas. Our data demonstrated that prophylactic injec-tion of DNA, either DNAhsp65 or DNAv, did not accelerate the development of clinical diabetes and, in fact, decreased the incidence of the disease [125]. The lower glycemic levels in mice previously injected with DNA were correlated with the exten-sion of insulitis. These animals presented significantly reduced prevalence of destructive insulitis, as well as a concomitant increase in the percentage of islets devoid of inflammation. This protective effect was far more accentuated in vaccinated than in vector-injected animals. Immunohistochemistry revealed that the protection mechanism involved Treg infiltration in local islets. Prior vaccine injection resulted in decreased levels of CD4+ and CD8+ T cells, as well as the concomitant appear-ance of CD25+ cells in the islet parenchyma. These changes were accompanied by locally decreased levels of TNF-α and increased levels of IL-10 [125].

More recently, the modulatory effects of this DNA con-struct were also evaluated in a rat model of EAE [Zorzella SF

et al., Manuscript Submitted]. The injection of vaccine or vector in Lewis rats before EAE induction by inoculation of MBP did not affect the clinical parameters (weight and clinical score) typically used to characterize the clinical EAE development. However, both DNA preparations significantly decreased the intensity of inflammatory infiltration in the brain. This effect was associ-ated with a clear immunomodulatory effect characterized by a decreased production of IFN-γ and IL-10 by cells from secondary lymphoid organs.

A common finding in these three autoimmune models was the protective effect associated with the empty vector. This activity was, in a general way, less expressive than the one imparted by the vaccine and was attributed to the existence of inhibitory sequences. Two antagonistic sequences have been identified in DNA derived from pathogens. The so-called immuno-stimulatory sequences exist in most pathogen DNA, activate the TLR9 pathway and induce strong Th1 responses, and are, therefore, used as adjuvants in different immunotherapeutic approaches [126]. On the other hand, inhibitory or immunoregu-latory DNA sequences neutralize immunostimulatory sequence activation via the TLR9 pathway [127]. Inhibitory sequences exist in many species, such as viral DNA, mutated CpG sequences



and repeat TTAGGG motifs in mammalian telomers [128–131], but their detailed mode of action is unclear so far. Therefore, further investigation of immunoregulatory DNA sequences and their influence on immune cells is expected to highlight TLR9 modulation to target these pathways for the treatment of diseases with inappropriate TLR9 signaling. However, studies on the functional impact of inhibitory ODN available so far are mainly limited to TLR9 signaling in murine models. The presence of immunoinhibitory CpG motifs in the hsp65 gene from M. leprae is not yet established. Moreover, by analysing regulatory phenotypic markers, including CD4+CD25hiCD103+

and CD4+CD25hiCTLA-4+, we observed that these cells were induced or activated in vaccinated animals [Santos-Junior RR et al.,

Manuscript Submitted], even though the specific T-cell epitopes recognized upon DNA hsp65 vaccination are still unknown.

In addition to these studies, comprehensive screening for autoimmune responses in mice vaccinated with DNAhsp65 did not indicate any tissue lesions different from vector- or saline-injected animals in 16 different organs (anti-DNA antibodies were also not found), even in the presence of cross-reactive anti-bodies against human Hsp60 protein [Lima DS et al., Manuscript

Submitted . Our most recent results from DNAhsp65 vaccination in nonhuman primates also reinforce the safety of this construct [Silva CL, Pers. Comm.].

The results obtained in experimental models of autoimmunity, infectious diseases and cancer strongly suggest that:

The high homology of the protein encoded in this vaccine •(Hsp65) with its mammalian counterpart (Hsp60) does not trigger or worsen autoimmunity;

Hsp65 posses immunomodulatory effects that can be used •against different illnesses;

Hsp65 is safe enough for submission to clinical trials.•

We recently participated in a Phase I clinical trial to estab-lish the safety and preliminary efficacy of DNAhsp65 in tumor immunotherapy. The potential use of M. leprae Hsp65 as an anti-cancer molecule has already been previously demonstrated [113]. The trial was conducted in 21 patients with squamous cell car-cinoma of the head and neck. All patients had unresectable and recurrent squamous cell carcinoma of the head and neck after standard therapies, with life expectancies of 3 months or longer. Toxicity was evaluated according to version 3.0 of the

National Cancer Institute Common Terminology Criteria for Adverse Effects. None of the patients demonstrated laboratory data compatible with autoimmune disease, and the most com-mon adverse events observed in some of the patients were edema and pain, which could be considered a natural consequence of the injection procedure. Although all patients were in an immu-nocompromised state, four exhibited increased peri pheral blood mononuclear cell proliferation. Moreover, reduction of tumoral mass was observed in four patients, of which two were still alive 3 years after the conclusion of the therapy [132]. In spite of the absence of an specific immune response against M. leprae Hsp65, including IgM or IgG antibodies or T-specific cells secreting IFN-γ in blood mononuclear cells, we observed some degree of immunostimulation in two of the patients with more favorable outcomes who became purified protein derivative positives after DNAhsp65 immunization [133].

This study was the first to test Hsp65 of mycobacterial origin in cancer patients. Moreover, the lack of autoimmune reactions, along with no increase in antibody or IFN-γ reaction against human Hsp60 and the clinical benefit observed in some patients, warrants further confirmatory studies designed to evaluate DNAhsp65 effects in a large number of patients [132,133].

Expert commentary The outstanding amount of promising results obtained with DNA constructs in autoimmune experimental models led us to believe that it is only a matter of time before they are used in the control of autoimmunity. Clinical trials with the most relevant autoimmune pathologies are mandatory. Strategies based on the association of DNA vaccines and classical immunossupressive therapies are warranted and expected in the near future. The most important obstacle in this field is probably the expand-ing, but still incomplete, understanding of the DNA vaccines themselves. Much work is necessary for improving biosafety and efficacy in humans.

Five-year view Increased scientific knowledge will probably result in safer and more efficient use of DNA vaccines. Determination of the real contribution of B cells and DCs to the immunopatho genetic mechanisms underlying immunity to self components is impor-tant, as are many details related to Treg cells, including subset characterization, optimization of activation protocols to induce

Key issues

DNA vaccines are among the most promising tools for immune intervention, owing to their immunomodulatory activity for prophylaxis • and therapy of a broad array of diseases.

Technologies for DNA inoculation and delivery, specifically those that target the nuclei of specific cells, are continually improving. •

The use of DNA vaccines against different autoimmune diseases with promising results suggests that they will become a health • resource for those conditions.

Nonetheless, the molecular events mediating DNA uptake, expression and degradation, as well as several biosafety aspects, have • been neglected.

Better characterization of the contribution of other cell populations, including B cells and dendritic cells, in autoimmune diseases is • expected to improve the engineering of DNA vaccines for these conditions.



their activation and expansion and also their modulation by envi-ronmental agents, are still waiting for a better understanding. Finally, studies focusing on patients themselves, in order to deter-mine their genetic or environmental propensity for autoimmunity, could eventually be used to design prophylactic protocols.

AcknowledgementsWe are grateful to the students and technicians of our laboratories for their great contributions to this research.

Financial & competing interests disclosureCelio L Silva is part of Farmacore Biotechnology Ltd. Our group has received grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and the Millenium Institute REDE-TB from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. The authors have no other relevant affiliations or financial involvement with any organiza-tion or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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AffiliationsCelio L Silva •The Centre for Tuberculosis Research, Department of Biochemistry and Immunology, Medicine School of Ribeirão Preto, University of São Paulo, CEP 14049-900, Brazil; and Farmacore Biotechnology Ltd., Ribeirão Preto, São Paulo, Brazil Tel.: +55 163 602 3086 Fax: +55 163 602 3238 [email protected]

Vânia LD Bonato •The Centre for Tuberculosis Research, Department of Biochemistry and Immunology, Medicine School of Ribeirão Preto, University of São Paulo, CEP 14049-900, Brazil

Rubens R dos Santos-Júnior •The Centre for Tuberculosis Research, Department of Biochemistry and Immunology, Medicine School of Ribeirão Preto, University of São Paulo, CEP 14049-900, Brazil; and Department of Clinical Analyses, Faculty of Pharmaceutical Sciences, São Paulo State University, Araraquara, São Paulo, CEP 14801-902, Brazil

Carlos R Zárate-Bladés •The Centre for Tuberculosis Research, Department of Biochemistry and Immunology, Medicine School of Ribeirão Preto, University of São Paulo, CEP 14049-900, Brazil [email protected]

Alexandrina Sartori •Department of Microbiology and Immunology, Biosciences Institute, São Paulo State University, Botucatu, São Paulo, CEP 18618-000, Brazil

recent advances in dna vaccines for autoimmune diseases

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