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Human Vaccines & Immunotherapeutics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/khvi20

Non-contact helium-based plasma for delivery of DNA vaccines ab

b

ac

d

Richard J. Connolly , Taryn Chapman , Andrew M. Hoff , Michele A. Kutzler , Mark J. ab

Jaroszeski a

& Kenneth E. Ugen

ae

Center for Molecular Delivery, University of South Florida; Tampa, FL USA

b

Department of Chemical and Biomedical Engineering, College of Engineering, University of South Florida; Tampa, FL USA c

Department of Electrical Engineering, College of Engineering, University of South Florida; Tampa, FL USA d

Department of Medicine, Drexel University College of Medicine; Philadelphia, PA USA

e

Department of Molecular Medicine, Morsani College of Medicine, University of South Florida; Tampa, FL USA Published online: 16 Aug 2012.

To cite this article: Richard J. Connolly, Taryn Chapman, Andrew M. Hoff, Michele A. Kutzler, Mark J. Jaroszeski & Kenneth E. Ugen (2012) Non-contact helium-based plasma for delivery of DNA vaccines, Human Vaccines & Immunotherapeutics, 8:11, 1729-1733, DOI: 10.4161/hv.21624 To link to this article: http://dx.doi.org/10.4161/hv.21624

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Research Paper

Special Focus Research Paper

Human Vaccines & Immunotherapeutics 8:11, 1729–1733; November 2012; © 2012 Landes Bioscience

Non-contact helium-based plasma for delivery of DNA vaccines

Enhancement of humoral and cellular immune responses 1 Center for Molecular Delivery; University of South Florida; Tampa, FL USA; 2Department of Chemical and Biomedical Engineering; College of Engineering; University of South Florida; Tampa, FL USA; 3Department of Electrical Engineering; College of Engineering; University of South Florida; Tampa, FL USA; 4Department of Medicine; Drexel University College of Medicine; Philadelphia, PA USA; 5Department of Molecular Medicine; Morsani College of Medicine; University of South Florida; Tampa, FL USA

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Keywords: DNA vaccine, helium plasma, CTL responses, antibody responses, HIV

Non-viral in vivo administration of plasmid DNA for vaccines and immunotherapeutics has been hampered by inefficient delivery. Methods to enhance delivery such as in vivo electroporation (EP) have demonstrated effectiveness in circumventing this difficulty. However, the contact-dependent nature of EP has resulting side effects in animals and humans. Noncontact delivery methods should, in principle, overcome some of these obstacles. This report describes a helium plasma–based delivery system that enhanced humoral and cellular antigen-specific immune responses in mice against an intradermally administered HIV gp120-expressing plasmid vaccine (pJRFLgp120). The most efficient plasma delivery parameters investigated resulted in the generation of geometric mean antibody-binding titers that were 19-fold higher than plasmid delivery alone. Plasma mediated delivery of pJRFLgp120 also resulted in a 17-fold increase in the number of interferon-gamma spot-forming cells, a measure of CD8+ cytotoxic T cells, compared with non-facilitated plasmid delivery. This is the first report demonstrating the ability of this contact-independent delivery method to enhance antigen-specific immune responses against a protein generated by a DNA vaccine.

Introduction Non-viral vector-based DNA, that express specific proteins, has been used for vaccination and therapeutic purposes.1,2 Limitations on delivery and subsequent expression of DNA have, among other factors, lessened the effectiveness and enthusiasm for DNA vaccines.3 Generation of optimized DNA expression plasmids, as well as development of delivery enhancement methods, have been evaluated as methods to increase the efficacy of DNA vaccines.4-7 Electroporation (EP) is among a number of delivery enhancement modalities evaluated in recent years. This electric pulsebased delivery method has been successful in increasing expression and enhancing immune responses against proteins generated by DNA.7-10 This technique has been utilized to successfully deliver DNA to tumors in animals and humans.8,11 However, the contact-dependent nature and resulting side effects of the technique have limited wide adoption of this technology.12,13 Therefore, there has been an impetus to develop contact-independent methods for DNA delivery that function in a manner similar to EP. This report presents a study utilizing a contact-independent delivery method found to enhance humoral and cellular immune responses induced by a DNA vaccine. The method decouples the applicator from the target tissue by using “cold” helium plasma

as the delivery stimulating medium.14,15 Similar to EP, plasma mediated delivery is thought to function by creating an electric field at the treatment site sufficient to cause transient destabilization of cell membranes. A constant deposition of ions from the plasma source establishes an electric field at the treatment site. Proof of this concept was first demonstrated when a high frequency plasma discharge was used in vitro to deliver DNA encoding a fluorescent protein to neural cells.16 Building upon these results, a direct current ionic discharge generated in atmospheric air, known as corona discharge, has been used to enhance in vitro delivery of chemotherapeutic agents to melanoma cells.17 More recently, a piezoelectric spark applicator has proven capable of enhancing plasmid DNA delivery in vivo by transmitting pulsed high electrical currents through air to a treatment site.18 The study presented in this report, uses a direct current helium plasma capable of transiently disrupting cell membrane integrity without adversely effecting cell viability.15 Plasma mediated delivery has been used to deliver tracer molecules in vitro, as well as genes in vivo.14,15,19 Specifically, a previous short report demonstrated the enhancement of antibody responses against a DNA vaccine delivered by helium plasma.19 However, in that study, the ELISA methodology was performed with a protein not 100% homologous to the vaccinating antigen. As well, cellular

*Correspondence to: Richard J. Connolly and Kenneth E. Ugen; Email: [email protected] and [email protected] Submitted: 06/05/12; Revised: 07/20/12; Accepted: 07/25/12 http://dx.doi.org/10.4161/hv.21624 www.landesbioscience.com

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Richard J. Connolly,1,2,* Taryn Chapman,2 Andrew M. Hoff,1,3 Michele A. Kutzler,4 Mark J. Jaroszeski1,2 and Kenneth E. Ugen1,5,*

immune response against the expressed antigen were not assessed in that study. However, the study reported here documents the first use of the plasma mediated delivery technology to enhance the generation of both antigen specific cellular and humoral immune responses against a vaccine delivered by DNA. Results and Discussion Figure 1A shows the plasmid map for the DNA vaccine expressing the macrophage-tropic HIV-1 JRFLgp120 envelope glycoprotein (pJRLgp120).19,20 Figure 1B demonstrates delivery of helium plasma to mouse skin. Further descriptions of the helium plasma device, delivery parameters, and the gp120-expressing plasmid are described in “Materials and Methods.” Figure 2 shows the vaccination regimen used including the time points for vaccination and blood/tissue sampling.

Figure 2. Vaccination (Vax), bleed (sera) and tissue sampling schedule for the study.

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Figure 1. (A) DNA vaccine plasmid map of the pVAX-based CMV promoter-driven JRFLgp120 expression vector. (B) Method of helium plasma delivery to the skin of a BALB/c mouse. Plasma generator was operating at +8kV. The afterglow was captured with a 30 sec exposure.

Figures 3 and 4 show antibody binding and cellular immune responses respectively from analysis of mouse sera or spleen samples taken at day 91 (i.e., 7 d after the 5th vaccination). Figure 3 shows specific geometric mean antibody binding titer (GMT) values for sera binding to the JRFLgp120 protein as measured by enzyme-linked immunosorbent assay (ELISA), using the terminal bleeds from mice in the different treatment groups. The highest anti-JRFLgp120 GMT (GMT = 623,487) was obtained when the JRFL plasmid was delivered at 2 min with positive plasma. Specific antibody binding levels were 19-fold higher (significant at p < 0.05) than GMT values obtained with plasmid injection alone (GMT = 32,768). Likewise, the GMT value for the 2 min positive plasma delivery was significantly higher (p < 0.05) than values in all the other vaccination groups. The other plasma delivery condition groups tested resulted in antibody binding GMT values less than those observed in the 2 min positive plasma conditions, but values were significantly elevated in all other conditions (p < 0.05) compared with plasmid injection alone. These results suggest that the duration of plasma exposure might be more critical than excitation polarity for inducing enhancement of specific GMT antibody titers. This indicates that the enhancement of antibody responses occurred through delivery of pJRFLgp120 via helium plasma, with 2 min positive plasma delivery time mediating the most significant enhancement. Cellular immunity was assessed by measuring interferongamma (IFN-γ) spot-forming cells (SFCs) in the vaccination groups by an enzyme-linked immunosorbent spot (ELISpot) assay. Antigen-specific IFN-γ production by immune cells is widely accepted as an indication of a CD8 + cytotoxic T cell response.21 Figure 4 summarizes the results obtained from this analysis. The analysis indicated that plasma mediated delivery of pJRFLgp120 significantly (p < 0.05) enhanced the generation of IFN-γ SPC compared with plasmid delivery alone (SFC = 40). SFCs in the plasmid plus 2 min positive plasma were 17 times higher than in the plasmid-alone group (SFCs of 672 vs. 40), and induction of IFN-γ was significantly (p < 0.05) higher than values in other delivery groups. Compared with the 2 min positive plasma delivery conditions, the 2 min negative plasma was the next most potent group in eliciting IFN-γ SFCs, but the mean of the SFC values was not statistically elevated when compared with the other groups utilizing plasma delivery. Delivery of plasmid DNA with 0.5 min of plasma exposure did not result in significantly elevated IFN-γ positive SFCs. Helium plasma shows promise as a method for delivering plasmid DNA vaccines, but a definitive mechanism for delivery has yet to be elucidated. The hypothesis for the plasma effect is that

Figure 3. Enhancement of anti-JRFLgp120 antigen-specific humoral immune responses indicated by geometric mean antibody binding titers (GMT). Values above each column are the antigen specific binding GMTs. P only = pJRFLgp120; P + 0.5-minPP = pJRFLgp120 delivered by 0.5 min of a positive-polarity plasma stream; P + 0.5-minNP = pJRFLgp120 delivered by 0.5 min of a negative-polarity plasma stream; P + 2-minPP = pJRFLgp120 delivered by 2.0 min of a positive-polarity plasma stream; P + 2-minNP = pJRFLgp120 delivered by 2.0 min of a negative-polarity plasma stream. ★ indicates that the GMT for the particular group was significantly elevated (p < 0.05) compared with the P only group.

Materials and Methods Plasma delivery conditions. Construction and characterization of the plasma generator have been previously described.19 In the plasma conditions tested in this study, delivery times were either 0.5 or 2 min with a space of 1 cm between the generator and target tissue. These intervals were selected because they were found to significantly enhance expression of luciferase encoding plasmids. The difference between positive and negative plasma is the polarity of the electrical potential applied to the plasma generator to achieve ionization of the gas. Potentials of +8 kV (positive plasma) or -8 kV (negative plasma) were applied to the electrode of the generator to achieve ionization. To focus the plasma beam on the site of injection a loop of 22 gauge tinned copper wire was placed symmetrically around the bolus. This wire was connected to ground potential through a 1.5 GΩ resistive load. DNA plasmid and vaccination/delivery schedule. Six to ten week-old female BALB/c mice (NIH) were utilized in this study. All procedures involving animals were conducted in accordance with an approved University of South Florida Institutional Animal Care and Use protocol. Animals were prepared for vaccination and other treatment as described previously.19 The JRFLgp120-expressing DNA plasmid was constructed under the control of a CMV promoter. The HIV JRFL envelope glycoprotein was selected on the basis of the finding (unpublished

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Figure 4. Enhancement of anti-JRFLgp120 antigen-specific cellular immune responses measured by and interferon-γ (IFN-γ) enzymelinked immunosorbent spot (ELISpot). Values above each of the graph columns are mean number (± SE) IFN-γ spot-forming colonies (SPC) per 106 splenocytes. Spots indicate representative ELISpot wells from the pJRFLgp120 injection-only group as pJRFLgp120 + plasma delivery groups. NT Control = non-treated group. The other experimental group designations are as given in the legend for Figure 3. ★ indicates that the GMT for the particular group was significantly elevated (p < 0.05) compared with the P only group.

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a transdermal electric field is established by continuous deposition of electrically charged gaseous species on the epidermal skin surface. This electric field mediates the enhanced biological effect similar to the purported mechanism for EP. For EP, this is thought to be the induction of transient membrane defects, or pores, which allows the passage of exogenous molecules, including DNA, into the cytosol.15,22,23 It is also relevant to report that tissue histological analysis after helium plasma delivery failed to demonstrate any significant tissue damage (unpublished results). This supports previously reported observations of no visible skin damage following plasma treatment.14 Experimental results indicate that 2 min of plasma exposure correlates to the highest immunological responses, which could result from either an increase in total plasmid delivered, or from enhancement of local immune responses at the treatment site due to a mild and temporary inflammatory response. Factors such as these will be among the focus of studies aimed at elucidating the mechanism of plasma-enhanced DNA delivery. Future studies will focus on the effects of incremental increases in plasma treatment time to determine if intermediate times such as 1 min could produce more significant immunological responses. After an optimum treatment time is determined, experiments will focus on modulating plasmid doses to maximize cellular and humoral responses. Additionally, these studies will seek to understand why plasma polarity plays an integral role in enhancing antigen-specific immune responses. In summation, non-contact helium plasma appears to be effective in enhancing DNA vaccine mediated immune responses and may be a complementary method to EP delivery. As well, it could be an alternative to EP because of its non-contact delivery characteristics.

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GMTs for each of the vaccination groups are reported in this study. IFN-γ ELISpot assays were performed as previously described.30 ELISpot Millipore (Billerica, MA) 96-well plates were coated with an R&D Systems anti-mouse IFN-γ capture antibody and incubated for 24 h at 4°C. A total of 2 × 105 splenocytes from immunized mice were added per well of an ELISpot plate and stimulated at 37°C, 5% CO2, in the presence of R10 media (negative control), concanavalin A (positive control), or specific peptide (HIV-1 envelope, consensus) antigens (10 μg/ml). The HIV-1 envelope 15-mer peptides spanning the entire HIV-1 envelope consensus B subtype, overlapping by 11 amino acids, was acquired from the AIDS Reagent and Reference Repository. As a control, cells were stimulated with the CD8 + T cell epitope peptide encoding influenza A/PR/8/34 (H1N1) hemagglutinin (IYSTVASSL amino acids 518–526) synthesized by Immunodiagnostics. Following 24 h of stimulation, plates were washed and incubated for 24 h at 4°C with biotinylated antimouse IFN-γ antibody (R&D Systems). The plates were subsequently washed, streptavidin-alkaline phosphatase (R&D Systems) was added to each well, and incubated for 2 h at room temperature. Each plate was washed, then 5-bromo-4chloro-3' indolylphosphate p-toluidine salt (BCIP) and nitro blue tetrazolium chloride (NBT) (R&D Systems) were added to each well. Spots in each well were enumerated and were represented as IFN-γ SFCs per 106 splenocytes (CTL ImmunoSpot). Statistical analysis. Mean and standard deviations were calculated for each treatment group. Antibody data against JRFLgp120 were collected in duplicate using serial dilutions. Resulting antibody titers were analyzed at the 95% confidence level (p < 0.05) using analysis of variance (Kruskal-Wallis) combined with Dunn’s post-test analysis. ELISpot data presented in this report were collected from triplicate wells of pooled lymphocytes from each experimental group. Analysis between immunization groups of spots detected was performed with a two-tailed, paired Student’s t-test and yielded a specific p-value for each experimental group. This analysis also used a 95% confidence level (p < 0.05) to determine differences among the vaccination groups. Disclosure of Potential Conflicts of Interest

Richard Connolly, Mark Jaroszeski, and Andrew Hoff are inventors on patent applications involving the technology utilized in this study. Additionally, Mark Jaroszeski holds stock options in Inovio Pharmaceuticals, Inc., a company whose major emphasis is the development of electrical delivery technologies. None of the other co-authors of the work declare any conflicts of interest. Acknowledgments

The study described in this paper was supported, in part, by grant AI079706 from the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIAID. In addition, the work was also supported, in part, by a pilot grant from the Florida

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result) that this plasmid resulted in only moderate expression and immunogenicity under conditions of plasmid injection alone. Therefore, it was reasoned that the potential expression and immune enhancing effects of the delivery method would be more evident and measurable with this plasmid. Plasmid injections were performed in the shaved left flank skin with a 1 ml syringe and a 30 gauge needle (Becton Dickinson). Plasmids were injected intradermally with 100 μg of pJRFLgp120 in 50 μl physiological saline. Discharge exposure times of 0.5 and 2 min were selected based on other published and unpublished in vivo data. Each of the different vaccination conditions were applied to groups of 12 mice. The following vaccination groups were evaluated in this study: no treatment, i.e., not injected with plasmid nor exposed to plasma; 100 μg pVAX plasmid backbone only; 100 μg pJRFLgp120 only; 100 μg pJRFLgp120 plus 0.5 min positive plasma; 100 μg pJRFLgp120 plus 0.5 min negative plasma; 100 μg pJRFLgp120 plus 2 min positive plasma; and 100 μg pJRFLgp120 plus 2 min negative plasma. The protocol consisted of vaccinations on days 0, 14, 28, 42 and 84 and submandibular bleeds, to generate sera for ELISA analysis, 7 d after each treatment. This vaccination schedule is summarized in Figure 2. At day 91, mice in all groups were humanely euthanized. Spleens were immediately harvested and prepared for an IFN-γ ELISpot analysis. The experimental immunization schedule and specimen collection time points were selected based on other intradermal EP-delivered vaccines reported in the literature using mouse models as well as guinea pig models.9,24-29 ELISA and ELISpot analysis. To measure antibody titers, an indirect ELISA was performed as described previously.19 This assay involved coating 96-well plates (Immulon 4Hbx Plates FlatBottom, ISC Bioexpress) with 100 ng (2 μg/ml) of JRFLgp120 recombinant protein (eENZYME, LLC) in carbonatebicarbonate buffer (Sigma-Aldrich), and incubating for 24 h at 4°C. Plates were then blocked with 3% bovine serum albumin (Fisher Scientific) and 0.05% Tween20 (Fisher Scientific) in phosphate-buffered saline (Fisher Scientific) for 2 h at 25°C. Serum samples were added to plates in a series of 2-fold dilutions from 1/8,192 thru 1/1,048,576 diluted in blocking buffer, and allowed to incubate for 24 h at 4°C. Anti-mouse polyvalent immunoglobulins (G,A,M)-peroxidase antibody produced in goat (Sigma-Aldrich) were then added at a 1:10,000 dilution in blocking buffer and allowed to incubate for 2 h at 25°C. After each incubation step, wells were washed four times with 300 μl of a phosphate buffered saline solution containing 0.05% Tween20. To develop each well, a solution of 3,3'5,5'-tetramethylbenzidine dihydrochloride (Sigma-Aldrich) in phosphate citrate buffer (Sigma-Aldrich) was added and allowed to incubate at 25°C in dark conditions. After 15 min, development was stopped with 2 N sulfuric acid. Optical density of the wells was determined with a 450 nm filter on a plate reader (BioTek Synergy). Endpoint antibody binding titers (EPTs) for each mouse were determined and defined as the reciprocal of the most dilute titer that was at least three standard deviations higher than the mean OD450 nm of the non-treated control group. EPTs were then used to calculate GMTs by previously described methods. The

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Center of Excellence in Biomolecular Identification and Targeted Therapeutics (FCoE-BITT). We also acknowledge Dr. David Weiner and Dr. Jian Yan, from the University of Pennsylvania, for generously supplying the JRFLgp120 expressing DNA plasmid. Joseph Register of the University of South Florida provided