Current Gene Therapy - Volume 14, Issue 3, 2014

Conventional immunization approaches utilize live attenuated pathogens, inactivated organisms, recombinant proteins or polysaccharide antigens to induce protective immunity. Twenty years ago in a major breakthrough it was shown that immune responses could instead be elicited by injecting plasmid DNA encoding relevant vaccine antigens [1-3]. This heralded the start of DNA vaccination. DNA vaccines offer many potential advantages; including speed and simplicity of manufacture. Despite early hype, this technology has yet to yield approved human products although there are already a number of approved veterinary DNA vaccines suggesting human applications are only a matter of time [4]. It should be remembered that monoclonal antibodies took over 2 decades from initial discovery to final successful human application. By these standards DNA vaccine technology is still in its relatively infancy. Hence this special edition on DNA vaccines is timely to examine the state of the art in DNA vaccine technology. It is hoped this collections of papers will help address the perennial question asked on all long journeys, “are we there yet?” These papers convey a sense of the tremendous distance that DNA vaccine technology has come over the 20 years since its initial discovery. In particular, issues of DNA vaccine safety have by and large been satisfactorily addressed, leaving vaccine efficacy as the only real remaining challenge [5]. Despite the passage of time there is still a sense of excitement that surrounds the DNA vaccine field. These papers convey a willingness of those in the field to press on to solve the remaining challenges to bring DNA vaccines to the human market. This augurs well for the eventual success of DNA vaccine technology. A variety of key topics are covered by this collection. The excellent review by Jim Williams describes the state of the art in DNA plasmid design. It highlights just how far plasmid design has been advanced and explores how plasmids can be fine tuned for maximal protein expression. Kwilas et al., describe a novel delivery approach that uses a jet injector device to deliver the plasmid intramuscularly without the need for a needle. Interestingly this form of administration appears to also enhance plasmid expression and vaccine immunogenicity. Another area where there have been major advances is the area of DNA vaccine adjuvants. Capitani et al. demonstrate that plasmids encoding aggregation-promoting domains act as DNA vaccine adjuvants by triggering frustrated autophagy leading to caspase activation and apoptotic cell death. The induction of cell death is common to traditional vaccine adjuvants including alum and squalene oil emulsions [6], but poses safety risks as excess cell death may trigger unwanted side effects and even autoimmunity in susceptible individuals [7, 8]. No discussion of DNA vaccines would be complete without including electroporation as a method of enhancing plasmid expression. Davtyan et al. describe studies on electroporation settings to maximize delivery of an Alzheimer’s disease DNA vaccine encoding a β-amyloid epitope. Electroporation remains a potent tool for maximizing DNA delivery but with the downsides of inconvenience, cost and discomfort. Finally, Lucyna Cova examines the history of hepatitis B DNA vaccine development, describing the many challenges encountered along the way. This is a story that could easily be repeated for the many other DNA vaccines under development. I trust this collection of papers on current DNA vaccine research will convince the reader that the field of DNA vaccines is not dead, and in fact under the surface vigorous research and development efforts continue towards a key milestone which will be approval of the first human DNA vaccine. Considering the more than 20 years that monoclonal antibody technology had to spend in the wilderness before all their problems were solved and they became the pharmaceutical industry’s biggest success story, DNA vaccines may yet have their time in the sun.

Despite the existence of an effective prophylactic vaccine, chronic hepatitis B virus (HBV) infection remains a major public health problem. Because very weak and functionally impaired virus-specific immune responses play a key role in the persistence of HBV infection, the stimulation of these responses appears to be of particular importance for virus clearance. In this regard DNA-based vaccination has emerged as novel, promising therapeutic approach for chronic hepatitis B. This review provides an update of preclinical studies in animal models (mouse, chimpanzee, duck, woodchuck), which evaluated the ability of DNA vaccines targeting hepadnaviral proteins to induce potent and sustained immune responses in naïve animals and to enhance virus clearance and break immune tolerance in chronic virus-carriers. Different strategies have been developed and evaluated in these models to optimize DNA vaccine including genetic adjuvants, combination with antiviral drugs, prime-boost regimens and plasmid delivery. The delivery of DNA by in vivo electroporation appears to be of particular interest for increase of vaccine potency in both small and large animal models. Based on the promising results generated in preclinical studies, first clinical trials of DNA vaccines have been initiated, although effective therapy of chronic hepatitis B awaits further improvements in vaccine efficacy.

Background: DNA vaccines provide high tolerability and safety but commonly suffer from suboptimal immunogenicity. We previously reported that a plasmid vector (pATRex), encoding the DNA sequence for the von Willebrand I/A domain of the tumor endothelial marker-8 (TEM8) when given in combination with plasmid-encoded tumor antigens acted as a powerful molecular adjuvant enhancing immunity against breast and melanoma tumors. Aims: In the present study we addressed two unsolved issues; would the adjuvant action of pATRex extend to a DNA vaccine against infectious disease and, if so, what is the mechanistic basis for pATRex adjuvant action? Results: Here we show in a murine malaria vaccine model that co-administration of pATRex potentiates antibody production elicited by an intramuscular injection of plasmid encoding Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5). pATRex enhanced the B-cell response and induced increased IgG1 production consistent with TH2 polarization of the DNA vaccine response. To explore the mechanism of adjuvant action, cells were transfected in vitro with pATRex and this resulted in formation of insoluble intracellular aggregates and apoptotic cell death. Using a structural modeling approach we identified a short peptide sequence (α3-β4) within ATRex responsible for protein aggregation and confirmed that transfection of cells with plasmid encoding this self-assembling peptide similarly triggered intracellular aggregates, caspase activation and cell death. Conclusion: Plasmids encoding aggregationpromoting domains induce formation of insoluble intracellular aggregates that trigger caspase activation and apoptotic cell death leading to activation of the innate immune system thereby acting as genetic adjuvants.

DNA vaccines are a rapidly deployed next generation vaccination platform for treatment of human and animal disease. DNA delivery devices, such as electroporation and needle free jet injectors, are used to increase gene transfer. This results in higher antigen expression which correlates with improved humoral and cellular immunity in humans and animals. This review highlights recent vector and transgene design innovations that improve DNA vaccine performance. These new vectors improve antigen expression, increase plasmid manufacturing yield and quality in bioreactors, and eliminate antibiotic selection and other potential safety issues. A flowchart for designing synthetic antigen transgenes, combining antigen targeting, codon-optimization and bioinformatics, is presented. Application of improved vectors, of antibiotic free plasmid production, and cost effective manufacturing technologies will be critical to ensure safety, efficacy, and economically viable manufacturing of DNA vaccines currently under development for infectious disease, cancer, autoimmunity, immunotolerance and allergy indications.

DNA vaccines promote immune system activation in small animals and exhibit certain advantages when compared to conventional recombinant protein vaccines. However in clinical trials DNA vaccines are less effective in inducing potent immune responses due to the low delivery efficiency and expression of antigens. Currently, various delivery devices such as gene-guns, bioinjectors and electroporation systems are being used in order to increase the potency of DNA vaccines. However, the optimal delivery parameters are required and must be carefully set to obtain the highest levels of gene expression and strong immune responses in humans. The focus of this study was to optimize electroporation settings (voltage, pulse length, pulse intervals, and number of pulses), as well as the route of administration (intradermal vs. intramuscular) and dosage of the DNA epitope vaccine, AV-1959D, delivered by the BTX AgilePulseTM system. As a result, we have chosen the optimal settings for electroporation delivery using different routes of immunization with this vaccine, generating (i) robust antibody production to the B cell epitope (a small peptide, derived from β-amyloid), and (ii) strong cellular immune responses to Th epitopes (a small synthetic peptide and eleven peptides from various pathogens) incorporated into DNA vaccine platform.

Sin Nombre virus (SNV) and Andes virus (ANDV) cause most of the hantavirus pulmonary syndrome (HPS) cases in North and South America, respectively. The chances of a patient surviving HPS are only two in three. Previously, we demonstrated that SNV and ANDV DNA vaccines encoding the virus envelope glycoproteins elicit high-titer neutralizing antibodies in laboratory animals, and (for ANDV) in nonhuman primates (NHPs). In those studies, the vaccines were delivered by gene gun or muscle electroporation. Here, we tested whether a combined SNV/ANDV DNA vaccine (HPS DNA vaccine) could be delivered effectively using a disposable syringe jet injection (DSJI) system (PharmaJet, Inc). PharmaJet intramuscular (IM) and intradermal (ID) needle-free devices are FDA 510(k)-cleared, simple to use, and do not require electricity or pressurized gas. First, we tested the SNV DNA vaccine delivered by PharmaJet IM or ID devices in rabbits and NHPs. Both IM and ID devices produced high-titer anti-SNV neutralizing antibody responses in rabbits and NHPs. However, the ID device required at least two vaccinations in NHP to detect neutralizing antibodies in most animals, whereas all animals vaccinated once with the IM device seroconverted. Because the IM device was more effective in NHP, the Stratis® (PharmaJet IM device) was selected for follow-up studies. We evaluated the HPS DNA vaccine delivered using Stratis® and found that it produced high-titer anti-SNV and anti-ANDV neutralizing antibodies in rabbits (n=8/group) as measured by a classic plaque reduction neutralization test and a new pseudovirion neutralization assay. We were interested in determining if the differences between DSJI delivery (e.g., high-velocity liquid penetration through tissue) and other methods of vaccine injection, such as needle/syringe, might result in a more immunogenic DNA vaccine. To accomplish this, we compared the HPS DNA vaccine delivered by DSJI versus needle/syringe in NHPs (n=8/group). We found that both the anti-SNV and anti-ANDV neutralizing antibody titers were significantly higher (p-value 0.0115) in the DSJI-vaccinated groups than the needle/syringe group. For example, the anti-SNV and anti-ANDV PRNT50 geometric mean titers (GMTs) were 1,974 and 349 in the DSJI-vaccinated group versus 87 and 42 in the needle/syringe group. These data demonstrate, for the first time, that a spring-powered DSJI device is capable of effectively delivering a DNA vaccine to NHPs. Whether this HPS DNA vaccine, or any DNA vaccine, delivered by spring-powered DSJI will elicit a strong immune response in humans, requires clinical trials.

In vivo electroporation is one of the most efficient methods for introducing the nucleic acids into the target tissues, and thus plays a pivotal role in gene therapeutic studies. In vivo electroporation in rodent brains is often involved in injection of nucleic acids into the brain ventricle or specific area and then applying appropriate electrical field to the correct area. Better understanding of the progress of electroporation in rodent brain may further facilitate gene therapeutic studies on some brain disorders. For this purpose, we briefly summarized the advantages, the procedures and recent progress of transferring nucleic acids into the rodent brain using in vivo electroporation.

One of the major goals in the research of autoimmune diseases is to develop specific therapies to regulate the expression and function of gene products that could contribute to restoring tolerance to self-constituents and replace conventional systemic immunosuppression, which is associated with important undesired side effects. Although significant progress has been made on the understanding of the pathogenesis of autoimmunity, therapies for these ailments have not seen a change. During the last decade, different strategies such as pharmacologic or gene therapy modulation of heme oxygenase-1 (HO-1) and the administration of its metabolic product, carbon monoxide (CO), have been shown to display beneficial immunoregulatory and cytoprotective properties. In different experimental autoimmune conditions, such as Experimental autoimmune encephalomyelitis, type-1 diabetes and systemic lupus erythematosus, genetic or pharmacological modulation of HO-1, as well as delivery of CO have shown to ameliorate disease progression. Furthermore, it has been demonstrated that dendritic cell and monocyte function can be modulated by HO-1 and/or CO. In this article, recent data related to the immunoregulatory properties of HO-1/CO will be discussed, focusing on their potential therapeutic use to treat autoimmune diseases.

Gene-directed enzyme prodrug therapy (GDEPT) consists in targeted delivery to tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. One of the major limitations of this strategy in clinical application was the poor prodrug activation capacity of suicide gene. We built a highly efficient suicide gene capable of bioactivating the prodrug cyclophosphamide (CPA) by fusing a CYP2B6 triple mutant with NADPH cytochrome P450 reductase (CYP2B6TM-RED). Expression of this fusion gene via a recombinant lentivirus (LV) vector converted resistant human (A549) and murine (TC1) pulmonary cell lines into CPA-susceptible cell lines. We tested the efficiency of our GDEPT strategy in C57Bl/6 immunocompetent mice, using TC1 cells expressing the HPV-16 E6/E7 oncoproteins. In mice bearing tumors composed only of TC1-CYP2B6TM-RED cells, four CPA injections (140 mg/Kg once a week) completely eradicated the tumors for more than two months. Tumors having only 25% of TC1-CYP2B6TM-RED cells were also completely eradicated by five CPA injections, demonstrating a major in vivo bystander effect. Moreover, surviving mice were rechallenged with parental TC1 cells. The tumors regressed spontaneously 7 days after cell inoculation or grew more slowly than in control naive mice due to a strong immune response mediated by anti-E7CD8+T cells. These data suggest that combining the CYPB6TM-RED gene with CPA may hold promise as a highly effective treatment for solid tumors in humans.