Since the discovery of DNA vaccination as a novel technology to induce antigen-specific immune responses, different DNA plasmid delivery approaches can be grouped into two major types. The first type includes “chemical approaches”, relying on the chemical and biochemical interactions between DNA molecules and the target cells. With this type of delivery, DNA plasmids are dissolved in various solutions, with or without carrier polymers (in lipid-form or other chemical natures), and delivered by conventional intramuscular or intradermal needle injections, transdermally or through mucosal administration [4
]. The second type of DNA delivery includes “physical approaches”. With this type of approach, the delivery of DNA plasmids is based on various forms of physical forces, such as shock wave, high pressure gas and electrical pulse. In general, the physical approaches require special devices that can produce external forces, such as a gene gun or an electroporation device [3
]. There have been limited studies to directly compare these two types of DNA vaccine delivery approaches, but available data indicates that the physical method is more effective in eliciting higher immune responses in animal studies and that the type of immune responses elicited by these two types of delivery methods may also be different [15
The difference between the chemical and physical approaches is further highlighted by the results from early phase DNA vaccine studies in humans. DNA vaccines delivered by chemical methods, including those with facilitating chemical agents, have not been shown to be particularly immunogenic. In the first series of DNA vaccine studies in humans, a group of asymptomatic HIV-infected individuals who received a DNA vaccine encoding an env
gene and a rev
gene from the HIV-1MN
isolate via conventional intramuscular needle injection. While this study confirmed the safety of DNA immunization, no consistent change in CD4 or CD8 T lymphocyte counts or in plasma HIV concentrations was observed [62
]. In another study, it was found that although a DNA vaccine encoding the P. falciparum
circumsporozoite protein for malaria, could elicit low level antigen-specific CTL responses through conventional intramuscular injection [64
], measurable antibody responses were not detected in any of the 20 volunteers [65
]. On the other hand, gene gun delivery of a plasmid expressing hepatitis B surface antigen (HBsAg) in naive volunteers showed promising immune responses in humans [1
]. Three administrations of plasmid DNA by this approach resulted in the generation of seroprotective antibody levels in all 12 volunteers. Furthermore, all 12 volunteers showed cell-mediated immune responses to HBsAg post-administration, highlighting the ability of the physical delivery approach to generate broad-based immune responses. More recently, a phase I human study using gene gun technology examined the safety and immunogenicity of a DNA vaccine, pPJV1671, which expresses the HA from the H3 Panama strain of the human influenza virus in healthy adults [2
]. Volunteers in the 4μg dose group (highest dose) achieved the criteria on all three parameters required for licensure by the Committee for Proprietary Medical Products (CPMP) in the European Union (seroconversion, seroprotection and GMT) 21 days after a single vaccination. These data highlight the potential of DNA vaccines in humans, when delivered by a physical method.
The above results suggest that the ability of DNA vaccines to elicit an immune response in a host appear to greatly depend upon the route of administration (i.e., chemical vs. physical delivery). A high level immune response appears to rely on the ability of the DNA plasmids to efficiently enter targeted cells and not so much on the amount of DNA that is administered. DNA immunization via gene gun administration allows the DNA plasmids to penetrate directly into the cytoplasm [3
], presumably resulting in the DNA being processed by antigen presenting cells (APCs) and subsequently presented to T cells [68
]. The same holds true for any method that increases the ability of the DNA to enter directly into the intracellular environment rather than into the extracellular space, as is true for conventional needle delivery.
Recently, additional methods based on physical principles, such as electorporation (EP), have demonstrated increased immunogenicity when compared to the chemical delivery method via needle injection alone approach, as EP presumably creates transient pores in the cell membranes, and increases movement of the DNA into the cells due to an electrophoretic effect [69
]. The effectiveness of EP has been shown in a number of studies [34
]. The application of electroporation, regardless of the site of injection, should favor the transfection of a greater variety of cells, including APCs. Furthermore, as an additional mechanism, mild tissue damage, which may be induced by electroporation, could provoke an influx of APCs, induce danger signals, and enhance the release of antigen from injured cells, thereby increasing antigen presentation, and also possibly provide adjuvant effect [73
]. The chemical approach of delivering DNA with conventional needle delivery has a particular disadvantage in that the DNA is deposited into the extracellular space and subsequently needs to be taken up by the cells –a critical step, which so far has not been able overcome by any particular chemical carriers.
In the current study, we compared the relative immunogenicity of the two leading physical delivery methods: gene gun and electroporation, in both rabbit and mouse models. Our data show that both of these methods are highly effective in inducing antigen-specific immune responses, and both are more immunogenic than the conventional needle injection method. The difference in immunogenicity between these two physical approaches is somewhat limited. While the gene gun approach is significantly more effective in eliciting antibody responses in rabbits and only slightly more effective in mice, when compared to the EP approach, this advantage appears to disappear when multiple immunizations are given. On the other hand, EP may be more effective in inducing higher Th1-type antibody immune responses as shown in our study while GG predominantly induced Th2-type antibody immune responses as well documented in literature [75
]. This is interesting because both are “physical delivery methods” and raise the question of whether IM injection, even after electroporation, is the key factor in controlling these differences. In the current study, we did not directly measure secreted cytokines because it is not clear whether changes in the Th subtypes that would have been examined in mice would be valid in non-human primates or humans, the “real” targets for improved DNA vaccine delivery. With the increasing use of EP in large animal studies and the pending phase I clinical study in healthy human volunteers, a more complete understanding of antigen-specific T cell responses, including the unique profiles of cell-mediated immune responses related to the use of different DNA delivery methods, will be gained by using polyfunctional cytokine analyses.
EP delivery is clearly more effective than IM delivery in our study but the magnitude of increase was less than previously reported [34
], reasons for which there may be a number of factors. First, the immunogenicity of DNA vaccines is highly dependent on individual antigens. The HA antigen is known to be highly immunogenic and since the HA gene insert used in the current study is codon optimized, the immunogenicity of the HA DNA vaccine is further improved [45
]. Other factors which influence the immunogenicity of DNA vaccines irrespective of whether or not GG or EP is used include the choice of DNA vaccine vector and the design of the testing antigen as part of the antigen engineering process [77
]. In combination, these factors may improve the immunogenicity of the DNA vaccine included in the current study which may result in a smaller difference observed between IM and EP deliveries.
In the current study, a caliper electrode model electroporator was used. Currently, there is no standardized technology in EP. There are at least 3–4 different major models in the field. The results learned from the current report may not completely represent the results from other models of electroporators. Future studies may need to overcome the commercial barrier and conduct parallel studies to examine the safety and efficacy of different models of electroporators, especially in humans using the same antigens.
While, in the current study, we utilized an ELISA to measure antibody responses to vaccination, other assays such as hemagglutinination inhibition (HI) would have been a more useful measure of protective antibodies against influenza. However, the main goal of the current study was to examine the immunogenicity, rather than the protective qualities of the different vaccination approaches and given that the same antigen is used, the levels of protective antibodies are usually proportional to the level of overall antibody response.
Previously, the relative immunogenicity of DNA vaccine delivery methods by EP or GG was studied in an anti-tumor model in transgenic mice [61
] where the overall antigen-specific immune responses between these two methods were very similar. The only difference was that GG is less effective than EP in controlling the incidence and the growth of spontaneous tumors, however, there was no data to rule out a non-antigen-specific effect in this finding. Future studies should include the development of standardized protocols for EP and to compare the different EP models that are available in the field, especially for human clinical studies for vaccines against infectious diseases. Furthermore, since immune responses may differ dramatically with the use of this immunization method based on the innate properties of the antigen used, future studies should also examine the factors that determine the immunogenicity of various individual antigens when EP is used as the immunization method. The availability of more than one physical delivery approach in the field of DNA vaccine is a positive sign that DNA vaccine technology is maturing and proliferating before being accepted as a vaccination method for general use.