Introduction

Vaccination is the most cost-effective way to prevent disease. However, there are still many diseases for which no vaccine exists or for which the currently available vaccines are inadequate. DNA immunization, which entails the administration of DNA encoding an antigen, may offer solutions in at least some of these cases. DNA vaccines use host cells as bioreactors for the production of proteins in vivo¹. By doing so, DNA vaccination mimics viral infection, with improved antigen presentation to the immune system relative to standard protein vaccines, and works more effectively at inducing cellular immunity as a result². Moreover, it offers these potential benefits without the safety and stability concerns associated with the administration of infectious agents.

DNA immunization has been effective in several mouse models³. To achieve significant levels of immunity in humans and large animals with DNA delivery methods often requires very high doses of plasmids and multiple boosts⁴. Clearly, there is a need to increase the potency of DNA vaccines in large mammals, including humans⁵,⁶. Even at high doses, DNA vaccines in humans still have failed to be efficacious⁶,⁷,⁸,⁹. Therefore, more potent forms of the DNA vaccines themselves and/or more effective means of delivery must be developed for the technology to realize its potential.

One reason for the inefficacy of DNA vaccines in humans could be inefficient cellular uptake of plasmid DNA and consequent poor gene expression. In vivo electroporation may allow increased gene expression by enhancing cellular uptake of plasmids by the application of short electrical pulses that transiently permeabilize cell membranes. Electroporation may also enhance nuclear uptake. It has been clearly shown that DNA delivery to muscle tissue of mice, followed by electroporation, strongly increases gene expression and immune responses elicited by DNA vaccines¹⁰,¹¹. Furthermore, when used for DNA delivery in the skin, electroporation amplifies gene expression with both intradermal injection¹² and topical administration¹³ of plasmids. Thus, it would follow that electroporation of skin should enhance DNA immunizations, and experimental results have confirmed that prediction in mice¹⁴.

One objective of this study was to explore whether the use of needle-free surface electroporation would significantly enhance gene expression and immune responses to a DNA vaccine in pigs. Another objective was to minimize the invasiveness of the immunization procedure. For this reason, needle-free injection of the DNA, using a BioJect device, was compared with conventional injection by needle and syringe. An additional incentive for using fluid jet injection over needle and syringe was the previous findings that jet injection was more effective for DNA immunization in monkeys and sheep¹⁵,¹⁶ compared to intramuscular needle administration of plasmid. Both the fluid jet and the needle methods of DNA injection were assessed with respect to their ability to induce an immune response with and without needle-free surface electroporation.

Numerous studies have shown that the most powerful property of DNA vaccines may be their ability to prime the immune system for responses to other vaccines¹⁷,¹⁸. We examined the effects of electroporation in the context of a DNA-prime/DNA-boost/protein-boost strategy to enhance immune responses in large animals and compared the responses with the responses following standard protein vaccination. This strategy resembled DNA-prime/protein-boost strategies previously used in nonhuman primates, which yielded outstanding results for both malaria¹⁹ and HIV vaccinations²⁰.

Results

Gene expression and effect of electroporation on the skin

To characterize gene expression of plasmid DNA delivered by intradermal needle or needle-free BioJect delivery, we used the luciferase and green fluorescent protein (GFP) reporter genes. First, we determined where the injected materials were localized in the skin by using Evans blue dye. Both the needle and the needle-free methods of injection delivered most of the dye to the dermis. Needle-free injection also deposited dye in the epidermis and the hypodermis and, thus, resulted in a broader distribution than that obtained by standard intradermal needle injection (data not shown).

To quantify the level of gene expression, we used the luciferase reporter gene. The luciferase assay results indicated that delivery by BioJect was significantly better than needle injections, with respect to the level of gene expression detected (Fig. 1).

Figure 1.

Electroporation improves gene expression levels, and intradermal jet injection is more effective than intradermal needle injection. Luciferase-encoding plasmid DNA was administered to abdominal porcine skin, and enzymatic activity was measured 48 h following administration. Gene expression levels are shown for intradermal BioJect (b.j.) and needle (i.d.n.) injections, without or with electroporation at the voltages indicated. Error bars represent SEM. Statistical differences were i.d.n. vs b.j. (^*P < 0.05) and i.d.n. vs i.d.n. plus electroporation (^*P < 0.05) by one-way ANOVA followed by Tukey's multiple comparison test.

Full figure and legend (41K)

Electroporation at 60 or 80 V significantly enhanced the level of expression following intradermal needle injection (Fig. 1). Similarly, BioJect delivery of plasmids followed by electroporation enhanced gene expression following a 70- or 80-V pulse but did not following electroporation at 60 V.

Examination of the location of gene expression using a plasmid encoding GFP supported the results of luciferase expression, with BioJect showing greater expression than intradermal needle injection and electroporation enhancing gene expression following both BioJect and intradermal needle injection (data not shown).

Following electroporation, there was superficial damage on the stratum corneum that increased with higher voltages. This superficial damage to the stratum corneum was transient and no longer detectable 1 week following electroporation. This observation supports the contention that topical electroporation is relatively safe and does not cause long-term scaring.

We performed histological examination of tissue sections to determine how voltage could influence gene expression. The results of histological examination (Fig. 2) showed that increasing the voltage increased the size of the lesion on the surface of the skin, as well as increasing the level of cellular infiltration consisting of macrophages in the dermis and epidermis of the skin. The voltages chosen for the DNA immunizations were voltages that enhanced gene expression with the minimal amount of tissue damage. Those voltages were 60 V with the intradermal needle injections and 70 V with the BioJect deliveries.

Figure 2.

Effect of electroporation on tissue damage and cellular infiltration. Skin tissues were collected 48 h following electroporation at 60 (A), 70 (B), and 80 V (C) and stained with H&E. Cellular infiltration of macrophages and neutrophils in the stratum corneum was found in (A). Moderate cellular infiltration of macrophages and neutrophils in the epidermis was found in (B). Severe cellular infiltration in the epidermis as well as the dermis was observed in (C). Bar represents 100 m.

Full figure and legend (163K)

Immune responses in immunized pigs

Four weeks after the primary immunization, no animals in any of the groups had any detectable immune responses to hepatitis B surface antigen (HBsAg). At 8 weeks following a second protein immunization, the two groups of animals immunized with a subunit vaccine injected either intramuscularly using a needle and syringe (the recommended route) or intradermally with the BioJect responded with antibody levels considered protective by the AUSAB assay (Table 1). Results indicated that the intramuscular injection of the conventional vaccine resulted in all animals, 5/5, responding, whereas only 4/5 animals responded to intradermal injection of the conventional vaccine using the BioJect (Table 1). At 8 weeks, following a secondary plasmid vaccination, no groups immunized by intradermal injection with or without electroporation had any responding animals as detected by the AUSAB assay. Groups immunized with plasmid administered using the BioJect or the BioJect with electroporation had 1/5 and 2/5 animals, respectively, responding with antibody levels considered protective by the AUSAB assay.

Table 1 - Responses in pigs to various hepatitis B vaccination protocols.

Full table

To assess the efficacy of DNA vaccination in priming the immune system, we boosted animals in all experimental groups with the HBsAg protein vaccine at week 8. Results in Table 1 demonstrate that even though immune responses were undetectable in most of the animals after two rounds of DNA immunization with respect to the AUSAB clinical assay, most animals responded rapidly to the protein boost. However, only DNA vaccines administered with electroporation and the HBsAg protein vaccines were significantly different compared to prebleed serum (Fig. 3). The fact that nearly all DNA-immunized animals responded within 2 weeks of the protein boost demonstrates an anamnestic response, since the protein vaccine alone did not induce detectable immune responses 4 weeks postimmunization. There were no statistical differences between the immunized groups at 10 weeks, when immune responses were determined by the AUSAB clinical assay.

Figure 3.

Immune responses to hepatitis B after the different DNA-prime/protein-boost immunizations. HBsAg antibody titers at 10 weeks (2 weeks after the protein boost) were determined using the AUSAB EIA and are the geometric means of five animals for all the groups except for the i.m.n. subunit group, which had four animals. Error bars are SEM. Statistical differences were determined using a one-way ANOVA followed by Tukey's multiple comparison test; ^**P < 0.01, ^***P < 0.001 vs prebleed.

Full figure and legend (43K)

To determine whether the experimental manipulations had an impact on the antibody isotypes generated, we analyzed the sera for the presence of anti-HBsAg IgG₁ and IgG₂ using an ELISA. Needle-free plasmid injection in combination with electroporation led to a more rapid induction of immune responses compared to other methods of plasmid delivery (Fig. 4). Fig. 4 demonstrates that in those animals mounting an early response, most produced primarily IgG₁ at 8 weeks. However, after being boosted with protein, a much more balanced response with approximately equivalent levels of IgG₁ and IgG₂ was evident.

Figure 4.

Antibody isotype responses elicited by hepatitis B immunizations. Hepatitis B-specific IgG₁ (A, D) and IgG₂ (B, E) titers at 8 (A, B) and 10 (D, E) weeks and IgG₁/IgG₂ ratios from responding animals (C, F) are shown; data represent individual animals at 8 and 10 weeks and the bars are the geometric means. Group 1 is BioJect DNA, Group 2 BioJect DNA + EP, Group 3 intradermal injection DNA, Group 4 intradermal injection DNA + EP, Group 5 BioJect administration of the subunit vaccine, and Group 6 intramuscular injection of the subunit vaccine. At week 8, prior to boosting with protein, the level of antibody (IgG₁) in the BioJect DNA plus electroporation group vs i.d.n. DNA plus electroporation (P < 0.001) was statistically different by one-way ANOVA followed by Tukey's multiple comparison test. There was no statistical difference between the levels of antibody (IgG₁) in the BioJect DNA + electroporation and the Engerix-B groups at 8 weeks by one-way ANOVA followed by Tukey's multiple comparison test.

Full figure and legend (115K)

Discussion

There is a critical need to improve the potency of DNA vaccines if DNA vaccines are to be used routinely for vaccination. Recently, electroporation was shown to enhance the potency of DNA vaccines in mice and rabbits¹⁰ as well as pigs²¹. However, intramuscular electroporation using two- and six-needle arrays are relatively invasive. Using a unique needle-free surface patch electrode, a significant enhancement of gene expression following intradermal plasmid administration was demonstrated. Furthermore, to eliminate the use of needles in the vaccine administration, a needle-free intradermal injection device combined with noninvasive electroporation was tested and it was found to be more effective than intradermal needle injection. It was shown that a totally needle-free delivery–electroporation system was the most effective way to administer DNA vaccines in the skin.

Luciferase gene expression experiments revealed that needle-free BioJect administration resulted in higher levels of gene expression than needle injection. This observation is in agreement with previous results showing that jet injections elicit higher gene expression levels than needle injections in rat and human skin²². One possible reason for this result may be that administration with BioJect more efficiently distributes plasmid in the tissue, as determined by gross visualizations of injection patterns with Evans blue dye and GFP expression. This corroborates recent research findings that larger fluid injection volumes enhanced gene expression in muscle tissues²³. Another possible reason for greater gene expression following delivery by the BioJect device could be the transient disruption of membranes by the jet streams. Such disruption would increase the ability of the plasmid DNA to enter the cells and resemble the role of electroporation, which permeabilizes the cell membranes through the formation of pores as a result of applying high-intensity electrical fields. This would explain why electroporation enhanced needle injection to a greater extent than jet injection. However, any cellular DNA uptake resulting from membrane disruption by the jets appears to be much less significant than the permeability increase offered by electroporation, because substantial gains were still seen from coupling electroporation with jet injection, relative to the use of BioJect alone.

Topical electroporation sets up an electrical field that is greatest at the surface of the skin and diminishes in strength with increased distance from the electrodes. Although electroporation enhances gene expression with both needle injection and BioJect administration, the electroporation voltage needed was higher in the case of the jet injection. The difference in depth of plasmid distribution in the skin by the two delivery methods may explain this observation. The BioJect device delivered liquid more deeply into the skin than the purposely shallow intradermal needle injection. This deeper delivery may require a greater electroporation voltage because a sufficient electrical field is required to permeabilize cells in deeper layers of skin. However, electroporation at higher voltages causes greater tissue damage, which can reduce gene expression, especially in the epidermis, seen in the GFP studies (data not shown). Increased gene expression in the dermis offsets this effect. These conclusions concur with those of Glasspool-Malone et al., who used invasive electrodes and showed that electroporation of skin changes the distribution of gene expression and concentrates it in the dermis²⁴.

Although the mechanisms by which electroporation enhances immunogenicity of DNA vaccines are not yet completely understood, the increase in antigen production associated with electroporation is likely important. Studies in mice showed that reduction of antigen expression decreased immune responses²⁵, and increases in antigen expression enhanced immune responses²⁶. The results here support these suggestions since electroporation enhanced both gene expression and immune responses. Increasing the effectiveness of a DNA vaccine requires raising the levels of antigen production and/or increasing antigen uptake by antigen-presenting cells (APCs) to heighten the immune response. Electroporation may serve to enhance the immune response not only by increasing antigen expression, but also by other effects on the electroporated tissue. The tissue damage, cellular infiltration, and inflammation caused by electroporation may trigger the release of cytokines that attract APCs to the exact site where increased levels of antigen are being produced. This explanation would resemble the mechanism of agents such as bupivacaine that damage muscle and thereby enhance the immune response when it is delivered prior to DNA immunization²⁷. Thus, the minor damage caused by electroporation could be advantageous for eliciting immune responses.

The determination of antibody titers by both AUSAB and conventional ELISA generally yielded similar results for the various animal groups. However, there were discrepancies for individual animals. These individual differences are explainable by the different parameters measured by each assay. AUSAB, in effect, measures levels of antibodies by sandwiching them between antigen and other antibodies. In contrast, the conventional ELISA less selectively measures levels of antibodies that bind at a certain affinity/avidity. Therefore, the observed discrepancy is expected.

The needle-free means of injection in combination with electroporation was more effective than needle injection at improving the kinetics of the immune response to DNA priming and boosting. These results are consistent with our luciferase assay results, which indicate that electroporation enhances gene expression, and needle-free delivery is more effective than needle delivery of plasmid DNA. Both delivery methods, each with electroporation, were about equally effective at producing an immune response after the protein boost. At 10 weeks all of the groups achieved a similar level of AUSAB antibody responses. This suggests that the potency of the prime does not determine the magnitude of the ultimate response, perhaps only the kinetics of the response. The conventional intramuscular route of administration of the HBsAg subunit vaccine was previously found to be superior to the intradermal route of administration²⁸. Antibody titers at 10 weeks support that result, but also offer the perspective that needle-free intradermal delivery may be comparable to conventional intramuscular delivery, in terms of its elicitation of an immune response. That result is quite promising given the much less invasive nature of intradermal BioJect delivery.

In a previous experiment, in which 1 mg of the identical HBsAg-encoding plasmid was administered in muscle using a needle, only 2/6 pigs responded by the AUSAB assay after two immunizations (data not shown); therefore there are no significant differences between DNA immunization with HBsAg plasmid in the skin and in the muscle²¹. However, electroporation with the six-needle array in muscle resulted in all six pigs responding to the HBsAg DNA vaccine by the AUSAB assay²¹. The likely reason electroporation in muscle was more effective than needle-free electroporation in the skin was that electroporation in muscle resulted in a 10-fold greater level of gene expression compared to BioJect in combination with needle-free electroporation as measured by luciferase.

In summary, these experiments demonstrate that the needle-free method of intradermal DNA-vaccine delivery offers advantages, including increased efficacy of gene expression and safety, over injection with needle and syringe. In addition, electroporation is quick and simple to apply and is tolerated well by animals. Clearly, the use of a more complicated regimen with a DNA vaccine administered with electroporation will not replace a conventional vaccine. However, there are many diseases for which the conventional approach has not been effective. These results are very encouraging for the development of needle-free vaccination methods and the development of DNA vaccines.

Materials and methods

Plasmids

The luciferase-encoding plasmid (pluc), under the control of the CMV promoter in the pMAS backbone²⁹, was a gift from Dr. Heather Davis (University of Ottawa, ON, Canada)²⁵. The plasmid encoding GFP under the control of the CMV promoter was obtained through Quantum Biotechnologies (Montreal, QC, Canada). The hepatitis B surface Ag expression plasmid (pHBsAg), under the control of the human elongation factor 1 promoter, with the first intron and the polyadenylation signal from human G-CSF cDNA in a pUC9 prokaryotic backbone, was previously described¹⁰ and was custom-manufactured by Elim (San Francisco, CA). The other plasmids were purified using Qiagen Endo Free plasmid kits (Qiagen, Mississauga, ON, Canada) according to the manufacturer's instructions.

Animals and immunization

Four- to six-week-old male and female outbred pigs weighing 20 to 40 lb were purchased from the Prairie Swine Center (University of Saskatchewan, Saskatoon, SK, Canada). Animals were housed and treated in compliance with the Canadian Council for Animal Care. Pigs were randomly assigned to six groups of five animals each. They were anesthetized with halothane prior to DNA injection and electroporation. Group 1 received 250 g pHBsAg in 100 l 0.1 M phosphate-buffered saline (PBS) by a dermal BioJect B 2000 needle-free injection device (Bioject, Inc., Portland, OR) at each of two sites/animal on the abdomen, for a total of 500 g pHBsAg. Group 2 was identical to group 1 except both sites were also treated with 70 V electroporation using a flat patch needle-free surface electrode, immediately following plasmid injection. Group 3 received 250 g pHBsAg in 100 l PBS by an intradermal injection at each of two sites/animal on the abdomen for a total of 500 g pHBsAg. Group 4 was identical to group 3 except sites were treated with 60 V electroporation, immediately following plasmid administration. Group 5 received 500 l commercial hepatitis B protein subunit vaccine (Engerix-B, SmithKline Beecham Pharma, Oakville, ON, Canada) injected intradermally with the Bioject device in two 250-l doses an the abdomen and group 6 was immunized with Engerix-B by an intramuscular injection using a needle. Pigs were boosted with the same injection conditions after 4 weeks. All treatment groups were boosted at week 8 with Engerix-B vaccine by intramuscular needle injection for all groups except group 5, which was injected intradermally with Engerix with the BioJect device as above (Table 2).

Table 2 - Experimental design for vaccine groups.

Full table

Electroporation

Electroporation was performed using the BTX ECM 830 pulse generator with the needle-free micropatch round electrode mounted on a handle (Model MP 35) (Genetronics, San Diego, CA) and applying six square-wave pulses at 60, 70, or 80 V, respectively, with pulse duration of 60 ms, pulse interval of 200 ms, and reversal of polarity after three pulses. Electroporation conditions were selected based on previous studies in mouse skin done at Genetronics.

Luciferase expression and assay

Plasmid DNA encoding luciferase was injected intradermally with the use of a 26-gauge needle or a BioJect device (Dermal BioJect, BioJect, Inc.), followed by electroporation with various voltages. Intradermal needle injection was tested with no electroporation and with electroporation at voltages of 60 and 80. Bioject delivery was tested with no electroporation and with electroporation at voltages of 60, 70, and 80.

For each injection, a dose of 100 g of pluc²⁵ in 100 l PBS was administered into the shaved abdomen skin of the animals. The luciferase-encoding plasmid was injected at eight standard sites, and electroporation was applied to four of those sites. Forty-eight hours after the administration of the plasmid, each injection site was sampled with an 8-mm-diameter punch biopsy at a depth of approximately 8 mm.

Skin was homogenized in 500 l lysis buffer (Promega, Madison, WI) with a Polytron homogenizer (Brinkmann Instruments, Rexdale, ON, Canada) to produce protein extracts. Luciferase activity in the protein extracts was determined using a luciferase assay system (Promega). On a Packard Picolite Luminometer (Packard Instruments Canada Ltd., Mississauga, ON), the bioluminescence of each 500-l sample was counted for 30 s and recorded as relative light units (LUs). Untreated or PBS-injected tissues were used to determine the background luminescence levels.

Histological examination of skin

Forty-eight hours following intradermal injection by needle or BioJect of 100 l PBS alone or 100 g pluc in 100 l PBS followed by electroporation with 60, 70, or 80 V, skin tissues were taken and fixed in 10% formalin. Tissues were processed routinely and embedded in paraffin wax, and 4-m sections were stained with hematoxylin and eosin (H&E).

GFP gene expression

To analyze the localization of gene expression, 100 g of a plasmid encoding GFP in 100 l PBS was administered by intradermal injection or Biojector, in combination with electroporation (60 V for intradermal injection and 70 V for Biojector administration). Twenty-four hours after administration the injection site was biopsied using an 8-mm punch. Skin samples were frozen in liquid nitrogen and stored at -70°C until they were sectioned. Skin samples were cut transversally with an IEC Minitome Microtome Cryostat (Damon, Needham, MS) into 7-m sections. Sections were examined for GFP-expressing cells and were photographed with an Olympus AH2-RFL microscope using blue fluorescent light.

Measurement of humoral responses

At various time points after immunization, serum was obtained by centrifugation of the blood collected from anesthetized animals to measure if animals had immune responses that would be considered to protect against the disease. Anti-HBsAg antibodies were quantitatively measured using the AUSAB EIA Clinical Diagnostic Kit, and quantification in milli-international units per milliliter (mIU/ml) was performed in parallel with the AUSAB Quantification Panel, according to the manufacturer's instructions (Abbott Laboratories, North Chicago, IL). In humans, an antibody level >10 mIU/ml is considered to be protective for hepatitis B³⁰.

Anti-hepatitis B IgG₁ and IgG₂ isotypes were also identified by ELISA as follows. Immunlon 2 ELISA plates (DYNEX, Chantilly, VA) were coated with HBsAg (BioDesign International, Saco, ME) (1 g/ml in 20 mM Na₂CO₃) overnight at 4°C. Plates were washed with phosphate-buffered saline–Tween (PBST) (PBS, 0.05% Tween 20; Sigma Chemical Co., St. Louis, MO). Serum was diluted in diluent (PBST, 0.5% gelatin) (Sigma) 20-fold, followed by serial 4-fold dilutions, and incubated overnight at 4°C. Plates were washed six times in PBST. Porcine IgG₁ and IgG₂ isotypes were detected using specific mouse anti-porcine antibodies (Serotec, Hornby, ON, Canada). Following incubation at room temperature for 1 h, plates were washed six times in PBST. Anti-mouse IgG₁ biotinylated antibodies (Caltag, Toronto, ON, Canada), diluted in diluent, were added and incubated for 1 h at room temperature. Plates were washed six times in PBST, and streptavidin–alkaline phosphatase (Jackson Immuno-Research Labs, West Grove, PA) was added to the plates and incubated for 1 h. The alkaline phosphatase activity was measured by the conversion of p-nitrophenol phosphate (Sigma). The absorbance was read after 15 to 20 min at 405-nm wavelength on a plate reader (Bio-Rad, Hercules, CA).

Statistics

Differences between groups were analyzed using Prism GraphPad statistical software (GraphPad Software, Inc.) using a one-way ANOVA, followed by Tukey's multiple comparison test.

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Acknowledgements

We thank the animal care staff at VIDO for the care and handling of the animals, Dr. Dorothy Middleton for help with histology, Marlene Snider and Donna Mahony for technical assistance. We also thank Lorna Kocian for editing the manuscript. BioJect, Inc., Genetronics, Inc., and the Canadian Institutes of Health Research provided funding for this project. Shawn Babiuk is a recipient of a Natural Sciences and Engineering Research Council industrial graduate scholarship. Lorne Babiuk is a holder of a Canada Research Chair in vaccinology.