Genetic Immunization with Plasmid DNA Mediated by Electrotransfer

Abstract

The concept of DNA immunization was first advanced in the early 1990s, but was not developed because of an initial lack of efficiency. Recent technical advances in plasmid design and gene delivery techniques have allowed renewed interest in the idea. Particularly, a better understanding of genetic immunization has led to construction of optimized plasmids and the use of efficient molecular adjuvants. The field also took great advantage of new delivery techniques such as electrotransfer. This is a simple physical technique consisting of injecting plasmid DNA into a target tissue and applying an electric field, allowing up to a thousandfold more expression of the transgene than naked DNA. DNA immunization mediated by electrotransfer is now effective in a variety of preclinical models against infectious or acquired diseases such as cancer or autoimmune diseases, and is making its way through the clinics in several ongoing phase I human clinical trials. This review will briefly describe genetic immunization mediated by electrotransfer and the main fields of application.

Introduction

I n the early 1990s, administration of a naked DNA plasmid encoding an antigen was shown to elicit both humoral (Tang et al., 1992) and cellular (Ulmer et al., 1993) immune responses. Since then, numerous publications have reported the effectiveness of DNA immunization in a variety of preclinical models, including both infectious (either viral, bacterial, or parasitic) or acquired diseases, such as cancer (reviewed in Kutzler and Weiner, 2008; Rice et al., 2008; Mariana et al., 2010) or autoimmune diseases (Xue et al., 2011) and allergies (Chua et al., 2009). Although DNA vaccination outcomes in small animals are robust, they were at first quite ineffective in nonhuman primates and in early clinical trials. Possible explanations were that the level of protein expression was insufficient to elicit a strong and full immune response due to poor DNA delivery methods or that antigen expression from the plasmid was too low. Consequently, a number of new methods have been developed in the last 10 years to increase DNA uptake efficiency in order to increase antigen expression. Among the nonviral innovative methods, electrotransfer (or in vivo electroporation) has proven to be one of the most efficient means for in vivo gene delivery, particularly in the skeletal muscle (Trollet et al., 2006) where high levels of protein expression can be achieved, and more recently in the skin, to a lesser extent, reviewed in Gothelf and Gehl (2010). In addition to the delivery methods, great improvements have also been made in the design and optimization of the encoding eukaryotic expression plasmid (regarding both the bacterial backbone and the expression cassette) in order to increase the level and duration of antigen expression, and its immunogenicity. Also, improvements have been made in the use of adjuvants aimed at a better stimulation of the immune system and in the set-up of immunization protocols. All these works resulted in a better understanding of the DNA immunization process, which is now efficient enough to allow the licensing of three veterinary products and the first promising human clinical trials. This review will focus on recent advances in DNA immunization mediated by electroporation.

DNA Immunization

The principle of immunization is to induce the generation of memory T and B cells and the presence of neutralizing antibodies in the serum following the injection of a foreign antigen (either a whole organism or proteins). The observation that direct in vivo gene transfer of recombinant DNA resulted in expression of protein in situ led to the development of DNA vaccines. Introducing the gene encoding a protein directly into the skin (Tang et al., 1992) or muscle (Ulmer et al., 1993) of an animal elicits an immune response. In fact, this plasmid-based vaccine injection is an attractive approach because it provides several advantages over current vaccines (mainly live-attenuated pathogens, bacteria and viruses, or recombinant proteins). Plasmid DNA can be manufactured in a very cost-effective manner (ultrapure DNA preparation on a large scale is much easier than protein production and uses the same protocol regardless of the plasmid), and it is currently possible to order clinical grade good manufacturing practices (GMP) plasmids from suppliers. Plasmid DNA can be stored with relative ease (no need for a “cold chain” to maintain the efficiency of the vaccine because it is thermally stable), and there are none of the safety concerns associated with live-attenuated vaccine or pathogen or with viral vectors (particularly, no or very little DNA integration was found). Another advantage is that plasmid DNA does not induce any adaptative immune response against the vector, making it possible to re-administer the vaccine in contrast to most viral vectors. Finally, DNA immunization can trigger both a humoral and a cellular immune response. The organism will produce the antigen itself, inducing the immune response and thereby acting as a “bioreactor” (Van Drunen Littel-van den Hurk et al., 2004). It is furthermore possible to combine several plasmids to raise an immune response against multiple antigens as recently shown in the case of a smallpox DNA vaccine in nonhuman primates (Hirao et al., 2011).

The two most preferred target tissues for DNA vaccines are the skeletal muscle and the skin, the latter because of a high number of antigen-presenting cells (APC), the Langerhans cells (Nicolas and Guy, 2008). It is believed that transfecting more APCs will result in a stronger immune response. However, although the skeletal muscle is not considered the most efficient vaccination route mainly because myotubes, which are not APCs, are transfected, it increasingly appears that this organ plays an active role in immunity (Marino et al., 2011) and is efficient in raising a full immune response.

The mechanism by which plasmid DNA administration triggers an immune response is far from fully elucidated (partly because it is very complex, but also because most studies have been performed on small animals and results may be different in humans) and will not be described here. What is currently known is very well documented in several reviews (Kutzler and Weiner, 2008; Rice et al., 2008; Liu, 2010). Briefly, it appears that the immune response can be generated in two ways: either by direct transfection of some resident professional APCs in the muscle or the skin or by a cross-priming mechanism in which the antigen synthesized by myotubes or keratinocytes would be somehow released (by direct secretion or antigen-derived peptides released from necrotic or apoptotic bodies) and transferred to professional APCs, resulting in both a humoral and a cytotoxic T lymphocyte (CTL) response. With “naked” DNA immunization, high titers of neutralizing antibodies can be obtained in small animals, but because of low or poorly reproducible gene transfer efficiency, multiple immunizations of high DNA doses were often required to achieve modest responses in larger animals at first (Babiuk et al., 2003), particularly in primates (Widera et al., 2000). Early clinical trials were also very disappointing. The reasons for the lack of DNA vaccine efficacy in large animals and primates and in the first human clinical trials are not well understood, but it seems reasonable to think that a too low and highly variable in vivo production of the antigen resulting in insufficient immune stimulation was involved. Indeed, it has been demonstrated that the level of the immune response is directly related to antigen expression level (Lee et al., 1997; Kirman and Seder, 2003). This could come from an inefficient uptake of DNA by cells in the muscular tissue and from too low antigen expression from the plasmid. Therefore, it appears that raising a potent immune response depends on two components: an efficient DNA delivery method and an optimized plasmid allowing the highest antigen expression. In the last few years, research has focused on these two aspects and has led to important improvements in gene delivery efficiency and protocols, as well as plasmid optimization.

DNA Electroporation

Technical improvements in gene delivery methods have definitely proven critical in the renewed interest in genetic immunization. Several techniques have been developed, including cationic lipid or polymeric formulations (O'Hagan et al., 2006), tattooing (Van den Berg et al. 2009), and gene gun (Rottinghaus et al., 2003), but the most efficient to date seems to be in vivo electroporation. The nonpermeable lipophilic cell membrane controls the exchange of molecules between the cytoplasm and the external medium. Only a few hydrophobic molecules are able to cross the lipid bilayer and others can enter via specific transporter systems, but most of the hydrophilic molecules are unable to enter the cell. However, since the initial reports by Neumann et al. (1982), the use of electricity to mediate the delivery of molecules to cells in vitro is now a routine technique: by applying short and intense electric pulses, it is possible to transiently permeabilize the cell membrane, facilitating the entry of any molecule. This technique was subsequently used on living animals in the early 1990s. The first relevant in vivo application, electrochemotherapy, was demonstrated by cellular uptake into tumors of the antibiotic and chemotherapeutic agent bleomycin (Mir and Orlowski, 1999). Since then, the use of electric pulses to mediate plasmid DNA transfer became a rapidly emerging and promising technique, in vivo DNA electroporation or electrotransfer. Electrotransfer is based on injection of a DNA solution into a targeted tissue, followed by the application of a defined set of electric pulses to the tissue. The delivery of electric pulses enhances DNA uptake, resulting in a 100- to 1000-fold increase in gene expression (Bettan et al., 2000) (in comparison with naked DNA administration). This technique can be applied to any tissue, but the skeletal muscle is particularly suitable, mainly because it constitutes a large and easily accessible volume of tissue in which DNA electroporation is very efficient. Muscle fibers have a long lifespan because they are post-mitotic, allowing long-term expression (more than a year) in transfected cells (in the absence of regeneration due to injury or cytotoxic immune response (Mir et al., 1999). The skeletal muscle is made up of thousands of cylindrical muscle fibers bound together by connective tissue through which blood vessels and nerves run. This constitutes an abundant blood vascular system (Lu et al., 2003), and skeletal muscle is therefore able to produce secreted proteins with functional post-translational modifications, even of the size of a monoclonal antibody (Perez et al., 2004), that can easily reach the blood circulation (Goldspink, 2003). This has allowed multiple therapeutic approaches to be considered, such as direct gene transfer for cancer and muscle disorders, DNA vaccination, and systemic delivery of therapeutic proteins (Heller et al., 2005; Trollet et al., 2008; Heller and Heller, 2010; Hojman, 2010).

In the context of DNA immunization, electrotransfer greatly increases the potential of DNA vaccines since it increases antigen expression level by several orders of magnitude, resulting in an enhanced immune response towards the transgenes (Bachy et al., 2001), including in large animals (Otten et al., 2004; Scheerlinck et al., 2004) and in nonhuman primates (Otten et al., 2004). DNA immunization in a tissue rich in dendritic cells (e.g., the skin) leads more towards a Th2-type immune response, whereas intramuscular DNA immunization results in more of a Th1 humoral response (Harrison and Bianco, 2000). However, different results have been observed in a later study (Vandermeulen et al., 2007) showing that DNA electrotransfer into skin, muscle, and ear pinna all result in elevated interferon (IFN)-γ secretion and low interleukin (IL)-4 secretion, as well as a marked decrease of the IgG1/IgG2a ratio, thus indicating a Th1 shift in comparison with protein immunization, which displayed more of a Th2 profile. Another use of intramuscular DNA immunization is the production of a high titer antibody antiserum against a particular antigen, often a toxin. While increased transfection efficiency and concomitant increased antigen expression may explain the increased immune response in animals treated with DNA injection and electrotransfer (Widera et al., 2000), another reason for the efficacy of this technique is linked to the damage and inflammation induced in muscle cells by the electric field, causing the release of “danger signals” that create a local inflammatory context and recruit immune cells to the immunization site (Scheerlinck et al., 2004; Chiarella et al., 2008; Liu et al., 2008). Therefore, electroporation would act as an adjuvant for the antigen (Grønevik et al., 2004), enhancing the immune response, and would be favorable for maintaining immune memory (Tsang et al., 2007). The sum of these data illustrates the efficacy of DNA electroporation in eliciting a strong and full immune response, and this technique may greatly help to overcome the problem of scaling up from small animal experiments to clinical applications.

Plasmid Design

As already stated, the strength of the immune response is correlated to the level of the antigen expression and to the long-term antigen production (Miller et al., 2004). Besides the efficiency of the delivery technique, DNA immunization also requires the use of an optimal eukaryotic expression plasmid allowing high levels of expression for the longest period in vivo, and the highest immunogenicity, particularly in primates and humans. Therefore, careful design of the plasmid is essential. Basically, a plasmid is composed of a bacterial backbone and a transcription unit. Both components may have importance in the level of expression, and many works report modifications aiming at improving final antigen production and immunogenicity. The transcription unit usually comprises distinct genetic elements: promoter, cDNA sequence, translation termination codon, mRNA cleavage, and polyadenylation signal. The promoter is of critical importance and should ideally be strong, constitutive, and active in a wide range of cell types. Many promoters have been used for gene expression in mammalian cells (Makrides, 2003), such as Rous sarcoma virus, SV40, EF1-α, and human ubiquitin, but the most commonly used remains the human cytomegalovirus (hCMV) promoter (Foecking and Hofstetter, 1986), which is one of the strongest eukaryotic promoters and is found in most commercial vectors.

The level of expression may be enhanced by the presence of an intron, which was shown in the case of EF1-α intron inserted downstream of the CMV promoter (Kim et al., 2002). Besides the promoter, the transcription unit should also contain a proper transcription termination site and polyadenylation signal. The latter is important for mRNA stability and can enhance translation (Gray and Wickens, 1998). Several are used, including mouse β-globin or SV40 early transcription unit, with the most widely used being the bovine growth hormone sequence. The stop codon should allow proper translation termination by avoiding read through, which would result in an oversized antigen product and possible misfolding. It is common to add double stop codons in the cDNA sequence, preferably with a purine in the fourth position.

Engineering the coding sequence is one of the most effective strategies to improve antigen expression. In particular, codon optimization should be carefully considered: although all organisms share the same degenerated genetic code, they do not use synonymous codons at the same frequency. The use of synonymous codons is correlated with the respective abundance of the corresponding tRNAs. Therefore, using codons matching to low frequency tRNAs will result in a less efficient translation. Codon optimization consists of retaining the natural amino acid sequences but using the preferred codons for the targeted organism, resulting in enhanced antigen production and immune response (Doria-Rose et al., 2003; Frelin et al., 2004). This is particularly true when working with native sequences from organisms showing a very different codon bias as compared with mammals. This was recently illustrated in the case of the botulinum neurotoxin A: the difference between Mus musculus and Clostridium botulinum codon usage was 28.8% according to the Graphical Codon Usage Analyser (http://gcua.de/, Geneart). It has been observed that re-engineering the gene dramatically improved expression after transfection of B16 melanoma cells, and the antibody titer was increased 10-fold in mice after a single DNA electroporation immunization when using this optimized sequence (Trollet et al., 2009). In addition to codon optimization, the expression can also be enhanced by adding a Kozak consensus sequence surrounding the start codon. This sequence GCC(A/G)CCauG favors the right positioning of the ribosome to the start codon during translation initiation (Kozak, 2002).

Finally, the cellular localization and eventual extracellular secretion of the antigen might be a crucial parameter in controlling the magnitude of the immune response, due to the influence of the antigen presentation. For example, adding an endoplasmic reticulum retention signal fused to the antigen enhances the CTL response (Isagi et al., 2009). Several studies have also indicated that a secreted form of the antigen leads to a higher immune response and more neutralizing antibodies (Geissler et al., 1999; Ashok and Rangarajan, 2002). This can be obtained by fusing a signal sequence, usually at the 5′ end of the cDNA sequence. Commonly used signal sequences come from erythropoietin, tissue plasminogen activator, albumin, or immunoglobulin (Makrides, 2003). Interestingly, in the case of the botulinum neurotoxins, there was no difference in the total antibody titers between the nonsecreted and the secreted forms of the same antigen, but the neutralizing titers showed much higher levels in the latter case after a single immunization, resulting in a much stronger protective effect versus a challenge (Trollet et al., 2009). The neutralizing titer was even higher after a heterologous boost with the corresponding recombinant protein, a so-called prime/boost regimen. In immunization protocols, prime/boost consists of combining different methods of administration for the same antigen: for example, a prime with a DNA vaccine, followed by a boost with the corresponding recombinant protein or a viral vector encoding the same antigen. It is unclear why, but this regimen often increases the potency of a vaccine (Liu, 2010).

The bacterial backbone of the plasmid might also be of importance. A hallmark of vertebrate genomes is that cytosines within a CpG dinucleotide sequence are modified by a methyl group bound to the 5′ position of the cytosine ring (DNA methylation). Furthermore, CpG dinucleotides are underrepresented in mammalian genomes, a phenomenon referred to as “CpG suppression.” In contrast, bacterial DNA shows the expected frequency of CpG dinucleotides and is not methylated. The consequence of these two main differences is that mammalian cells recognize bacterial DNA, including plasmid DNA, as foreign. Some bacterial motifs (usually 5′-Pu-Pu-CpG-Pyr-Pyr-3′) are immunostimulatory and can stimulate Toll-like receptor 9 (Klinman et al., 1997), resulting in activation of macrophages, natural killer (NK) cells, monocytes, and dendritic cells (Krieg, 2002), and may therefore be used as adjuvants to increase the immune response against the antigen. However, the ability of these sequences to increase the plasmid DNA vaccines efficiency is still not clear (Liu, 2010) although CpG oligonucleotides do boost the immune response against protein antigens (Klinman et al., 2009).

Adjuvants

In addition to delivery techniques and plasmid design, the immune response can be enhanced or modified by the use of adjuvants. Adjuvants can either increase the global immune response or promote more of a humoral or cellular immune response, for example, by improving APC targeting or cross-presentation or by inducing local production of immunomodulatory cytokines. These adjuvants can be peptide or protein sequences fused to the antigen in the same way as was described for signal sequences, or additional molecular adjuvants co-administered with the DNA vaccines. As examples, fusing a weak lymphoma antigen to a strong immunogen derived from the tetanus toxin resulted in a dramatic increase in anti-antigen antibody and protection against lymphoma (reviewed in Rice et al., 2008). More recently, fusion of the antigen to a polyhistidine-Hsp70 sequence was shown to induce a strong antigen-specific CTL response after skin electroporation, probably through better APC targeting and innate immune system activation (Yamaoka et al., 2010). Other fusion sequences have been reported to enhance the immune response. In the case of the HIV gp120, fusion to an IgG provided a more effective protection against a chimera virus challenge (Shimada et al., 2010), and MIPα chemokine helped for APC targeting (Ruffini et al., 2010). Fusion of the host-derived component of complement C3d to the extracellular region of murine VEGFR-2 led to a significantly increased survival in mice melanoma and colon cancer models by inducing a strong humoral response (Xu et al., 2010), with similar findings in a bladder cancer model (Liang et al., 2010).

Molecular adjuvants usually co-administered as encoding plasmids with the antigen-encoding plasmid mainly consisting of cytokines or chemokines, with the aim of increasing immunogenicity. Some examples are interleukins (IL-2, IL-4, IL-7, IL-12, IL-15, IL-18), interferons, CD40L, ICAM-1, RANTES (reviewed in Abdulhaqq and Weiner, 2008), and HMGB1 (Muthumani et al., 2009).

Antibody Generation

Aside from the obvious potential of DNA immunization mediated by electrotransfer in the field of vaccination against infectious or acquired diseases, it should first be noted that it provides a unique laboratory tool to study antigenic properties of proteins and to generate either polyclonal or monoclonal antibodies. It offers several advantages, especially because it is faster than conventional protein immunization since only a plasmid is required and there is no need of recombinant protein production or purification. Recombinant protein production is often a long and limiting step, particularly in the case of low-yield proteins, unstable proteins, or in the case of large or membrane proteins that might prove difficult to produce. This is also true if the antigen needs posttranslational modifications and therefore has to be produced in eukaryotic cell systems, which might be laborious. Furthermore, because the antigen is produced in vivo, it is expected to have a more native conformation than a purified recombinant protein. Also, this technique takes advantage of the precision of molecular biology since the antigen can be defined at the single amino acid level, whereas recombinant proteins often carry His tags or short additional sequences necessary for purification. It is also possible to target the immune response towards a given epitope of interest by removing nonrelevant immunodominant epitopes. Finally, it allows to easily screen a large number of antigens for immunogenicity since it is easy to build and produce expression plasmids of research grade. This concept can be illustrated by a few examples.

GLTSCR2 is a human tumor suppressor candidate, which is very difficult to produce as a recombinant protein (Okahara et al., 2005). Its exact function remains largely unknown partly because no detection antibody has been developed; however, Okahara et al. (2005) were able to raise a rabbit polyclonal antibody against the N-terminal region of this protein suitable for immunocytochemistry analysis by skeletal muscle DNA electroporation.

A very interesting case is the prion protein (PrP) involved in neurodegenerative diseases: the recognition of the native conformation of PrP is an absolute prerequisite for anti-PrP antibodies to be used as therapeutic tools. In this work, genetic immunization by DNA electroporation in the muscle in mice was able to generate high amounts of antibodies recognizing the native PrP expressed on cell surface, unlike those obtained through classic protein immunization protocols (Alexandrenne et al., 2009). Furthermore, immunization with the human PrP which is a poor immunogen in mouse due to the high sequence homology between human and murine forms generated autoantibodies against the native murine PrP in wild-type mice, opening the way to studying the possible protective effects of autoantibodies against the PrP (Alexandrenne et al., 2010). This illustrates the most promising capacity of DNA electroporation to induce antibodies against a native form of an antigen, and to break the immune tolerance towards weak immunogens.

Finally, DNA electroporation can be useful in the generation of high affinity monoclonal antibodies. This was recently shown in the case of the retinol-binding protein 4, an important marker for kidney dysfunction. Recombinant protein immunization led to a poor immune response, and it was difficult to obtain monoclonal antibodies. Using a prime boost strategy, DNA immunization allowed raising high affinity antibodies against different epitopes of the protein. These antibodies were used to build an ELISA test and a gold fast test strip that might be suitable for a clinical use (Bian et al., 2010). Another example concerns house dust mite antigens, which are potent allergens and are involved in allergic diseases including asthma. Blo t 11 is one such allergen. It has a molecular mass of 102 kDa and is problematic to produce as a recombinant protein because it is very susceptible to degradation. It is therefore not possible to produce enough protein for conventional immunization of animals. This impediment was overcome by using intramuscular DNA immunization to produce monoclonal antibodies against Blo t 11 (Chua et al., 2008) as well as against Blo t 3, another mite antigen (Yang et al., 2003).

A monoclonal antibody against chemokine like factor 1 (CKLF1), a newly cloned human cytokine, was also generated by electroporation. Because CKLF1 is a highly hydrophobic protein, the purification of native CKLF1 was unsuccessful. Electrotransfer of a CKLF1-encoding plasmid into the skeletal muscle of mice (and therefore in vivo secretion of the protein) instead of the conventional protein immunization strategy overcame this problem and led to the generation of the desired monoclonal antibody (Chen et al., 2005).

In summary, these examples show that DNA electroporation is a helpful technique in the case of difficult-to-handle proteins in the generation of both polyclonal and monoclonal antibodies.

Autoimmune Diseases and Allergies

There is a growing interest in using DNA vaccines to treat allergies and autoimmune diseases (Chua et al., 2009; Liu, 2011). The aim is to modulate the immune response in a nonpathologic way. In the case of autoimmune diseases, DNA vaccines should induce tolerance rather than stimulate the immune responses to target antigens (Fissolo et al., 2010). In the case of allergy, DNA immunization would help to skew the immune response by generating a more Th1 response and inducing tolerance. Some interesting reports of DNA immunization have been published about type I autoimmune diabetes (Prud'homme et al., 2007) and multiple sclerosis (Fissolo et al., 2010). More recently, it was shown that electroporation of a DNA vaccine encoding the Pseudomonas exotoxin A and the co-immunostimulatory molecule B7-2 selectively decreased autoreactive Th1 cells in a rat model of collagen-induced arthritis, and had a potent antirheumatic effect comparable to the clinically used treatment with methotrexate (Xue et al., 2011). Another recent work reports that electroporation of a DNA vaccine encoding a tetanus toxin P30 Th epitope fused to the murine migration inhibitory factor (MIF) led to significant levels of auto-antibodies against MIF and protected mice from dextran sulphate sodium (DSS)-induced colitis (Ohkawara et al., 2011).

Cancer

Cancer represents a major field of application for DNA immunization. Electroporation-mediated gene transfer to fight cancer has been extensively studied in recent years, and direct intratumoral plasmid electrotransfer is a well-developed strategy for local production of therapeutic proteins; for example, cytokines encoding plasmid (Heller and Heller, 2010). A recent phase I clinical trial was performed and showed the safety, reproducibility, and clinical efficacy of this strategy, including cases of remission (Daud et al., 2008).

The discovery that some cancer cells are related to abnormal expression of tumor-specific or tumor-associated antigens has opened the way to the idea that an increased immune response against these tumor-specific antigens would help to fight against cancer. A variety of such antigens have been characterized (Bei and Scardino, 2010), which are considered excellent targets, particularly in melanoma, testicular cancer, or prostate cancer. However, these target antigens are weakly immunogenic, and the purpose of DNA immunization is to enhance a weak endogenous immune response and/or to break self tolerance.

The principles of DNA vaccination against cancer have already been well described (Rice et al., 2008; Stevenson et al., 2010). In this context, electroporation may help to increase the DNA vaccine potency. The first human clinical trials have already been performed. Particularly important results were obtained with the prostate-specific membrane antigen in the case of prostate cancer (phase I/II). The antigen was fused to a strong immunogenic epitope from the tetanus toxin (DOM) to increase its potency. The electroporation procedure was well tolerated and raised a humoral immune response in the majority of patients (Low et al., 2009). Furthermore, IFNγ-producing CD8⁺ cells were detected against the tumor antigen (Stevenson et al., 2010). This illustrates that electroporation is acceptable to patients and suitable for DNA vaccines against cancer.

Several other human clinical trials are currently active (clinicaltrials.gov) against prostate cancer with the prostate-specific antigen, melanoma (xenogenic tyrosinase), or leukemia (DOM-Wilm's tumor epitope fusion antigen), but the majority of work is still preclinical. Interesting results have been described against mammary carcinoma in mice using delta-like ligand 4 antigen, which targets angiogenesis. An immune response was obtained and orthotopic tumor growth was retarded (Haller et al., 2010), without side effects on wound healing. In another study, the HER2 receptor, which is a tumor-associated antigen expressed by several malignant cell types, was successfully administered by DNA electroporation in mouse mammary cancers (Curcio et al., 2008; Orlandi et al., 2011). The effect of the immune response was amplified when HER2 immunization was combined to a temporary regulatory T-cell depletion treatment, but only if the microscopic cancer was small enough (not palpable, Rolla et al., 2010). Other recent results describe the efficacy of the xenogenic chicken HSP70 antigen in a prime boost strategy for a canine transmissible venereal tumor (Yu et al., 2011), metalloproteinase MMP11 antigen in a colon cancer mouse model (Peruzzi et al., 2009), cancer testis antigen PASD1 fused to the tetanus toxin DOM epitope in a multiple myeloma mouse model (Joseph-Pietras et al., 2010), a human surviving epitope against the B16 melanoma mouse model after intradermal delivery (Lladser et al., 2010), or secreted telomerase combined with an adenovirus boost in a dog lymphoma model (Peruzzi et al., 2010).

Infectious Diseases

Most of the work on DNA immunization concerns infectious diseases. Indeed, two DNA vaccines are already licensed for veterinary use, one to protect horses against West Nile virus, and the other to protect salmon from infectious hematopoietic necrosis virus (reviewed in Liu, 2011). As previously discussed, DNA electroporation/electrotransfer represents a very suitable delivery strategy for vaccination purposes since it enhances the DNA vaccine potency to trigger both a CTL and a humoral immune response in conjunction with innate immune system stimulation.

The first human clinical trials using DNA electroporation have recently been completed, targeting serious and difficult to treat diseases such as HIV (ADVAX, NCT00545987) or HCV (CHRONVAC-C) for which a phase II trial is in its recruitment phase, following very promising Phase I results (NCT01335711). Some other trials are ongoing concerning HIV (PENNVAX, NCT01082692), H5N1 influenza strain (VGX-3400, NCT01184976), human papilloma virus (VGX-3100, NCT00685412), and malaria (EP-1300, NCT01169077) as reported in the http://clinicaltrials.gov/ (reviewed in Bodles-Brakhop et al., 2009; Sardesai and Weiner, 2011). The first human clinical results confirm that human DNA electroporation is safe and able to significantly elicit the immune response, as routinely shown in small animal preclinical studies.

In addition to these initial phase I clinical trials, a growing number of studies have been reported for other infectious diseases. Malaria is one of the most deadly diseases, responsible for over one million deaths per year, and there is no vaccine available yet. A DNA vaccine is thought to be a valid option, as long as conserved epitopes are found among the different strains of the Plasmodium falciparum parasite. The P. falciparum genome is different from mammalian genomes since it has an unusual 80% AT-rich content, which might lead to a low expression in mammalian cells. Therefore most studies have used codon-optimized sequence to screen for possible antigenic targets, with variable success. Codon optimization was found to enhance both humoral and cellular response in the case of the Pfs25 antigen (LeBlanc et al., 2008) versus the wild-type sequence, while only the humoral response was enhanced with PfCSP optimized antigen (Dobaño et al., 2009). To our knowledge, this is a very rare example of codon optimization failure. In a recent work, DNA electroporation was used to map the immunogenic properties of VAR2CSA, a candidate target against placental malaria. As VAR2CSA size is about 350 kDa, electroporation offers a unique tool for such a screening. A strong humoral immune response was obtained both in mice and rabbits vaccinated with a full VAR2CSA optimized sequence from a laboratory strain, and the resulting antisera totally inhibited binding of infected erythrocytes to its target. Remarkably, antibodies raised against a smaller part variant of VAR2CSA showed consistent inhibitory activity against several isolates originating from pregnant women (Bigey et al., 2011), thus identifying this region as a major target for a vaccine.

DNA electroporation has also proven a powerful tool to study and improve immunogenicity of viral antigens, particularly of the hepatitis B HBcAg antigen (Nyström et al., 2010) or HB-100 antigen (Kim et al., 2008) and hepatitis C virus antigens (Chen et al., 2011) in mouse models. Interestingly, the hepatitis C nonstructural region was able to elicit a strong cellular response in rhesus macaques and chimpanzees, which could be sufficient for preventive protection (Capone et al., 2006). Other encouraging results have recently been obtained in nonhuman primates against HIV (Luckay et al., 2007; Martinon et al., 2009; Hirao et al., 2010; Yin et al., 2010), Chikungunya virus (Mallilankaraman et al., 2011), anthrax (Livingston et al., 2010) or Venezuelan equine encephalitis virus for which protection from a pathogenic challenge was obtained (Dupuy et al., 2011). Some protection against challenge in small animals are also routinely achieved, as assessed by recent examples against H5N1 avian influenza virus (Chen et al., 2008; Laddy et al., 2008; Zheng et al., 2009), bovine viral diarrhea virus (Van Drunen Littel-van den Hurk et al., 2010), Schistosoma japonicum parasite (Dai et al., 2009; Zhu et al., 2010), or lymphocytic choriomeningitis virus (Shedlock et al., 2011).

Future Considerations

There is no doubt that genetic vaccination by plasmid electrotransfer represents a versatile and promising new avenue in the field of passive vaccination and/or active immunotherapy. This technique benefits from the conjunction of several unique advantages, such as sustained local production of the desired antigen, immune-adjuvant effect of the local electrotransfer-induced inflammation, as well as innate immune response stimulation by the plasmid backbone. In addition, this technology allows easy addition of immuno-adjuvant genetic sequences, as well as the association of different antigens by mixing different plasmids or by using plasmids bearing several expression cassettes or fusion genes corresponding to various dominant epitopic sequences.

Genetic vaccination by plasmid electrotransfer has also proved to be an interesting tool for mono- or polyclonal antibody production and antigen mapping, in cases in which other conventional techniques have failed or simply as a “first approach” technique due to its user friendliness. Therefore, we might expect a rapid generalization of genetic vaccination by plasmid electrotransfer. As far as veterinary or human applications are concerned, the development of genetic vaccination by plasmid electrotransfer is to be expected first for unmet medical needs, such as cancer or chronic infectious diseases, since the initial clinical trials show that the technique is safe and well tolerated. Research still has to be conducted concerning electrode devices and electric field characteristics, in view of widening the use of this technology to a large population in a broader field of applications. Several new devices are under development, including noninvasive skin-targeting electrodes that should reduce the associated pain (Donate et al., 2011). However, since the human immune system looks very different from that of mice, the success of DNA immunization will depend on designing clinical trials to obtain an optimal prophylactic or immuno-therapeutic response.

Footnotes

Acknowledgments

The authors are very grateful to Bérengère Jouy-Botte for reading and correcting the manuscript.

Author Disclosure Statement

No competing financial interests exist.

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