Molecular and cellular mechanisms of DNA vaccines

[Human Vaccines 4:6, 453-456; November/December 2008]; ©2008 Landes Bioscience

Commentary

Molecular and cellular mechanisms of DNA vaccines Cevayir Coban,1 Shohei Koyama,1 Fumihiko Takeshita,3 Shizuo Akira1 and Ken J. Ishii1,2,* 1Laboratory

of Host Defense; Immunology Frontier Research Center; World Premier Institute for Immunology; 2Department of Molecular Protozoology; Research Institute for Microbial Diseases; Osaka University; Suita, Osaka Japan; 3Department of Molecular Biodefense Research; Yokohama City University Graduate School of Medicine; Yokohama, Japan

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vaccines should elicit adequate and persistent humoral and/or cellular immune responses against the pathogen derived, protective antigen, which is mostly protein. In particular, in the last decade, researchers from the field of innate immunity have emphasized the importance of adjuvants in vaccine formulation as an essential component of successful vaccines. Modern microbiology, immunology and vaccine technology has opened new doors of understanding about the protection and treatment of not only infectious diseases, but also non-infectious diseases such as cancer and allergy. Here, we will comment on the recent advances on the mechanisms of action of DNA vaccines and their future implications for human use.

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Although DNA vaccines are already in use for treatment of some animal diseases, they suffer from lower immunogenicity in humans which limits their effectiveness. Thus, recent studies have been focused on strategies to improve the immunogenicity of DNA vaccines. However, there is little known about the molecular and immunological mechanisms by which DNA vaccines work. It has long been the central dogma that DNA vaccine immunogenicity can be attributed to its immunostimulatory CpG motifs acting as ‘a built-in adjuvant’, which is recognized by Toll-like receptor (TLR) 9, the sole receptor for CpG motifs. Recent research, however, has provided evidence for a new mechanism of action for DNA vaccines. It was reported that the adjuvant effect of plasmid DNA is mediated by its double-stranded structure, which activates TBK1-dependent innate immune signaling pathways in the absence of TLRs. Moreover, TBK1-signaling may delineate direct or indirect (cross) antigen presentation through distinct types of cells in vivo, critical for the induction of antigen-specific CD4+ or CD8+ T cells, respectively. This additional information about the mechanism of action of DNA vaccines will lead to improvements in their efficacy and safety.

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Key words: DNA vaccine, genetic vaccine, double-stranded B-form DNA, TBK1, interferon, adjuvant, TLR9, CpG motifs, innate immunity, infection

DNA Vaccine

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The DNA type of vaccine is a prime example of a modern type of vaccine (also called a genetic vaccine).2 A DNA vaccine is composed of a plasmid DNA that encodes the antigen of interest under the control of a mammalian promoter (i.e., CMV intron A) and can be easily produced in the bacteria.3 Once the plasmid DNA is administered in vivo, the encoded antigen is expressed in the host cells, and then processed and presented by antigen presenting cells such as dendritic cells. This most likely occurs in the draining lymph nodes, whereby eliciting both humoral and cellular immune responses. The ability to introduce antigen to the host immune system, thus enabling it to elicit strong Th1 type CD4+ T cells and CD8+ cytotoxic T cells, is a unique feature of a DNA vaccine which makes it distinct from conventional protein or peptide vaccines. As well, DNA vaccines are also free of the problems associated with producing recombinant protein vaccines (i.e., improper folding of targeted protein or high purification cost of protein purification), and they are also free of the possible risks associated with attenuated or inactivated vaccines which use infectious organisms. In addition, studies with DNA vaccines have shown that even after multiple immunizations, anti-DNA antibodies are not produced.4 Moreover, DNA vaccines can be easily manufactured and stored on a large scale. Since the early 1990s, DNA vaccines have been a promising strategy to develop immunity against several infectious diseases including HIV, tuberculosis, malaria, influenza and the newly emerging Ebola and SARS viruses.3,5-8 There have been remarkable successes achieved in most animal studies and clinical trials suggests that DNA vaccines are safe and well tolerated in humans.

Introduction

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Researchers are constantly looking for new vaccines which will provide protection from many infectious diseases. Probably the first “vaccine trial” was completed many centuries ago by Middle-Eastern populations to protect themselves against cutaneous Leishmaniasis through the inoculation of the pus from the active lesions into the skin of the foot of an uninfected infant to prevent further persistent scarring on important areas of the body such as the face.1 Despite the fact that these “early vaccines” had created the concept of “immunity,” it took quite some time for us to understand what was really happening in the body. From the last several decades of microbiology, immunology and medical research, we have learnt that successful *Correspondence to: Ken J. Ishii; Department of Molecular Protozoology; Research Institute for Microbial Diseases; Osaka University; 3-1 Yamadaoka; Suita, Osaka 565-0871 Japan; Tel.: +81.6.6879.8279; Fax: +81.6.6879.8281; Email: kenishii@ biken.osaka-u.ac.jp Submitted: 03/10/08; Accepted: 04/28/08 Previously published online as a Human Vaccines E-publication: http://www.landesbioscience.com/journals/vaccines/article/6200 www.landesbioscience.com

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Molecular and cellular mechanisms of DNA vaccines

Adjuvant Element of DNA Vaccine: The Dogma is Challenged

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However, the much lower immunogenicity of DNA vaccines has been observed in higher primates and clinical trials in humans with no apparent explanations. Because low immunogenicity has been the major deterrent towards the development of human DNA vaccines, several approaches have been investigated to improve it. Modifying the microenvironment of the vaccinated site by co-administration of genes, proteins or other immunologically active molecules are the most common strategies. For example, co-administration of genes encoding the co-stimulatory molecules cytokines and chemokines,9-11 genes that induce apoptosis,12 and genes encoding ligands for Toll-like receptors (TLRs) or their signaling molecules,13-16 have been shown to improve the immunogenicity of DNA vaccines. The addition of CpG motifs such as the TLR9 ligand in the plasmid17,18 or generating immunostimulatory RNA, such as double-stranded RNA by encoding replicon RNA,19 have also been demonstrated to enhance the immunogenicity of DNA vaccines. Moreover, targeting DNA towards proper antigen presenting cells (i.e., DEC-205-expressing DCs),20 combined vaccination with other modalities (e.g., ‘Prime-Boost’ immunization with DNA followed by a viral vector encoding the antigen or the protein antigen),21 and various immunization techniques and delivery systems of the DNA (i.e., electroporation, use of microparticles and tattoo-immunization)22-24 are some of the other approaches that have been undertaken. Despite these efforts to improve the DNA vaccine immunogenicity, we still do not know the precise cellular and molecular mechanisms by which a DNA vaccine works in the body. Specifically, what is the adjuvant element, how is innate immunity involved and how does the direct and indirect presentation of antigens proceed? Although we are still far from a complete understanding of these questions, it will be necessary to gather a substantial amount of information on these points into order to further improve DNA vaccine immunogenicity.

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Figure 1. Innate immune activation by DNA vaccine. Double stranded-B form of DNA and DNA vaccines utilize TBK1, whereas ssDNA and various RNAs utilize TLR9 and RIG-I/MDA5, respectively.

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Adjuvant has been “a little dirty secret” of immunologists25 and a bottle neck for modern vaccine development. This is mainly because we have gradually learned that successful vaccines need to contain a proper adjuvant element in order to activate the innate immune system to elicit immunogenicity to the protective antigen.26,27 The recent discovery of many innate immune system receptors such as the toll-like receptors (TLR),28 NOD-like receptors (NLR),29 and RIG-like receptors (RLR)30,31 have opened new avenues for the understanding of the effects and mechanisms of adjuvants that could improve vaccine development and design. It has been discovered that many of the innate immune system receptor ligands are protein, nucleic acid, lipid and carbohydrate in nature. Indeed, they have been used as adjuvants in vaccine trials for many years.27 Among them, modified lipid-A, poly-IC and CpG oligodeoxynucleotides (ODN) are examples that have been used frequently in animal models and in some clinical trials over the years.27 In the case of DNA vaccines, it has long been thought that the adjuvant element of a DNA vaccine is its specific sequence, the so called CpG motifs.6 It has been shown that plasmid DNA contains immunostimulatory CpG motifs, which are an unmethylated C followed by G and certain flanking sequences,32,33 a kind of “built in adjuvant” for DNA vaccines.34,35 In fact, the addition of many CpG motifs into a plasmid backbone has improved the immunogenicity of DNA vaccines.18,36,37 TLR9, expressed in the ER and/or the endosome of B cells and dendritic cells has been shown to be the only receptor for bacterial DNA and immunostimulatory CpG-containing synthetic DNA that elicits protective immune responses to many pathogens, allergic diseases and cancer when used as an adjuvant (Fig. 1).32,33 However, this story has been challenged by recent reports that TLR9-deficient mice showed comparable levels of antigen-specific IgG, IgG1 and IgG2a antibody responses and IFNγ and CTL responses as their wild-type counterparts.38,39 This data suggests that

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virus- or dsRNA-induced type I IFN production.44-46 Briefly, cytoplasmic helicases RIG-I and MDA5 recognize 5'-triphosphate RNA and dsRNA, followed by intracellular signaling through their sole adaptor IPS-1 as well as TRAF3, DUBA, TANK/NAP1/SINTBAD, which culminates in the activation of TBK1/IKK-i, which phosphorylates IRF-3 and IRF-7 (Fig. 1). However, no evidence has been reported as to whether TBK-1-mediated innate immune activation was involved in DNA-mediated innate immune activation.

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although the immunogenicity of DNA vaccines could be enhanced by additional CpG motifs in the plasmid backbone, the ‘basal’ adjuvant effects of DNA vaccines are mostly (if not entirely) independent of TLR9-recognition of CpG motifs. Therefore, what is the real adjuvant within a DNA vaccine? If there is any adjuvant element within DNA itself, what does this contribute to the immunogenicity of the vaccine as a whole? We now know that the double-stranded structure of DNA (most likely the right handed B-form) can stimulate the innate immune system independently of TLRs, instead signaling through non-canonical IKK and TANK-binding kinase-1 (TBK1).40 and that TBK1 is a key signaling molecule for DNA vaccine immunogenicity.41

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Figure 2. Direct versus cross priming of the adaptive immunity after DNA vaccination both require TBK1. DNA vaccine stimulates the induction of both antigen-specific B cells and CD4+ T cells in haematopoietic cells (i.e., dendritic cells) and CD8+ T cells in non-haematopoietic cells (i.e., muscle cells) through TBK1.

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TBK1-Dependent Innate Immune Activation by dsDNA

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Roles of TBK1 in TLR-Dependent and -Independent Signaling

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Discovery of the innate immune system receptors and their downstream signaling molecules together with adapter molecules has facilitated our understanding and interventions of the innate immune system. In the TLR-mediated signaling pathways, myeloid differentiation primary response gene 88 (MyD88) is found to be an essential adapter molecule for most of the TLRs except TLR3. Upon TLR-stimulation, transcription factor NFκB is activated through the canonical IκB kinase complex (IKK, composed of two catalytic subunits, IKKα and IKKβ) and MAP-kinases, which results in the production of pro-inflammatory genes including cytokines and chemokines. On the other hand, the other adapter molecule, toll/ IL-1R domain-containing adapter inducing interferon-beta (IFNβ) (TRIF) is necessary for TLR3 and TLR4-mediated signaling and induction of IFNβ and IFN-inducible genes. Specifically, TLR3 and TLR4 signaling and type I IFN production through TRIF activates the TRAF-family-member-associated NFκB activator (TANK) binding kinase 1 (TBK1), a non-canonical IκB kinase. TBK1 comprises another inducible IκB kinase (IKK-i, also known as IKK-ε) and these kinases directly phosphorylate interferon regulatory factor 3 (IRF3) and IRF7.42,43 In addition, recent evidence has suggested that TBK1/IKK-i-mediated type I IFN induction is not only restricted to TLR3 and 4, but is also involved in TLR-independent bacteria-,

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It has been shown that as well as bacterial or synthetic CpG-DNA, double stranded (ds) DNA derived from host cells in the B form (right-handed helical structure) and to a lesser extent, in the Z form (left-handed helical structure), could activate the innate immune system.40 When the double-stranded B-form of DNA is introduced into the cytosol by transfection, it activates fibroblasts, macrophages and dendritic cells to produce robust amounts of type-I interferons. This effect was independent of the CpG motif and TLR9, but heavily dependent on TBK1 (Fig. 1).40 It was subsequently confirmed by many groups that numerous microbial and host cell-derived DNAs are able to induce type-I IFNs in a TBK1-IRF3-dependent manner (reviewed in refs. 47 and 48). These findings have led investigators to search for a TLR-independent mechanism that senses the ds B-form of DNA. Recently, DLM-1/ZBP1 (also renamed later as DAI) was suggested to be a candidate receptor for dsDNA recognition.49 The authors showed in a mouse and human system in vitro that overexpression of DLM-1/ ZBP1 selectively enhances the DNA-mediated induction of type I IFN and other innate immune response genes. Further experiments using RNA interference also showed that RNAi for DLM-1/ZBP1 could inhibit specific gene induction upon stimulation by DNA.

TBK1, but not TLRs or DAI, Mediates DNA Vaccine Immunogenicity These findings raised a simple question as to whether TLR-independent immunogenicity of DNA vaccines is mediated through the dsDNA-induced innate immune activation via TBK1 or

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ZBP1 (DAI).27 In other words, since the adjuvant activity of plasmid DNA via TLR9 seems minimal in vivo, the ds B-form of plasmid DNA might be the critical adjuvant element necessary for the functioning of a DNA vaccine, especially when introduced into the cytoplasm and/or nucleus by transfection methods such as electroporation. We performed in vivo experiments to address this question.41 First, it was clearly shown using IFNaR2-deficient mice that the optimal DNA vaccine immunogenicity (antigen-specific T and B cell induction) required type-I IFNs. This was not attributed to TLR signaling as demonstrated by normal responses from MyD88/ TRIF-double deficient mice to DNA vaccines. Further analysis using TBK1-deficient mice, which are intact for TLR9-dependent innate immune responses, revealed that the DNA vaccine immunogenicity was completely dependent on TBK1 (Fig. 1). Moreover, bone-marrow transfer experiments indicated that TBK1-mediated signaling in haematopoietic cells, possibly dendritic cells, were mainly involved in the induction of both antigen-specific B cells and CD4+ T cells. On the other hand, a DNA vaccine which induced CD8+ T cells needed non-haematopoietic cells, probably DNA-transfected non-immune cells such as muscle cells. This suggested a distinct role for APC and transfected non-immune cells in direct versus cross priming of the adaptive immunity after DNA vaccination (Fig. 2). Finally, DNA vaccine immunizations of newly generated ZBP-1(DAI)-deficient mice showed no significant reduction of either innate or adaptive immune reponses to dsDNA or DNA vaccine in vivo, suggesting it plays a minimal role. One of the difficulties in understanding the sole adjuvant effect of dsDNA during DNA vaccination is that RNA could be generated during DNA vaccination and might act as an adjuvant by activating TBK1-dependent signaling. In fact, it has been previously suggested by many studies that ss- or dsRNA is recognized through TLR3-, TLR7/8-50 and RIG-I/MDA5,51 and by using the adapter molecules TRIF, MyD88 and IPS-1, respectively. However, these possibilities were later excluded using TRIF-, MyD88- and IPS-1-deficient mice when it was concluded that DNA vaccine induced immune responses were comparable to their WT counterparts.41 Recent advances clearly provide insights into the molecular and cellular mechanisms by which double-stranded structure, but not CpG motifs, is essential for DNA vaccine induced immunogenicity. This is believed to occur through the activation of type-I IFN-mediated innate immune activation resulting in the adjuvant effect to both antigen presenting cells and the DNA-transfected nonimmune cells to present the encoded antigen. In the future, research into the identification of the receptor for dsDNA and the mechanism of DNA vaccines may provide a boost to the medical community to develop novel vaccine adjuvants. Conversely, inhibiting innate immune activation through the TBK1 pathway would be beneficial towards the development of novel DNA-based gene therapies.

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Acknowledgements

We thank to Drs. T. Kawagoe and O. Takeuchi for their valuable inputs during these studies. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan.

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