Cutaneous Transfection and Immune Responses to Intradermal ...

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†Institute for Molecular and Immunologic Strategic Therapies, Rockville, Maryland 20850. ‡CytoPulse Sciences, Hanover, Maryland 21045. Received for ...
doi:10.1006/mthe.2000.0257, available online at http://www.idealibrary.com on IDEAL

ARTICLE

Cutaneous Transfection and Immune Responses to Intradermal Nucleic Acid Vaccination Are Significantly Enhanced by in Vivo Electropermeabilization Joseph J. Drabick,*,1 Jill Glasspool-Malone,†,1 Stella Somiari, Alan King,‡ and Robert W. Malone†,2 *Hematology–Oncology Service, Walter Reed Army Medical Center, Bethesda, Maryland 20307 † Institute for Molecular and Immunologic Strategic Therapies, Rockville, Maryland 20850 ‡ CytoPulse Sciences, Hanover, Maryland 21045 Received for publication June 7, 2000; accepted in revised form December 21, 2000

Naked DNA injection with electropermeabilization (EP) is a promising method for nucleic acid vaccination (NAV) and in vivo gene therapy. Skin is an ideal target for NAV due to ease of administration and the accessibility of large numbers of antigen-presenting cells within the tissue. This study demonstrates that in vivo skin EP may be used to increase transgene expression up to an average of 83-fold relative to naked DNA injection (50 ␮g DNA per dose, P < 0.005). Transfected cells were principally located in dermis and included adipocytes, fibroblasts, endothelial cells, and numerous mononuclear cells with dendritic processes in a porcine model. Transfected cells were also observed in lymph nodes draining electropermeabilized sites. A HBV sAg-coding plasmid was used to test skin EP-mediated NAV in a murine model. Analysis of humoral immune responses including immunoglobulin subclass profiles revealed strong enhancement of EP-mediated NAV relative to naked DNA injection, with a Th1-dominant, mixed-response pattern compared to immunization with HBV sAg protein that was exclusively Th2 (P ⴝ 0.02). Applications for these findings include NAV-based modulation of immune responses to pathogens, allergens, and tumorassociated antigens and the modification of tolerance. Key Words: gene therapy; nucleic acid vaccination; electroporation; electropermeabilization; skin; dendritic cell.

INTRODUCTION A growing number of recent reports have demonstrated that pulsed electrical fields may be used to safely and efficiently transfect tissues (1–3) as well as provide increased immune responses from muscle transfection with electroporation (4, 5). Adaptation of this transfection method for cutaneous applications may help realize some of the clinical opportunities in cutaneous gene therapy. During the past decade many researchers have verified that direct injection of “naked” plasmid DNA into skeletal muscle (6) and skin (7), or the bombardment of somatic cells with particles coated with plasmid DNA (8), results in

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These authors contributed equally to this work. To whom correspondence and reprint requests should be addressed at the MIST Institute, 308 Carr Avenue, Rockville, MD 20850. Fax: (240) 453-0860. E-mail: [email protected]. 2

MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy 1525-0016/01 $35.00

modest levels of transgene expression. These technologies are now commonly used for preclinical and limited clinical skin transfection research (9 –12). Much of this research is focused on the transient expression of antigens and/or immunomodulatory molecules to induce specific immune responses, termed nucleic acid vaccination (NAV). NAV was originally conceived as an application for naked DNA injection (6), but recently other technologies developed for gene therapy have begun to be adapted as well (13–15). It is difficult to compare the relative efficacy of different NAV methods. Comparative studies performed in a single laboratory often reflect the methods and expertise of the laboratory. During the preceding decade of NAV development, many different expression vectors (containing different immunostimulatory sequences), antigens, animal models, DNA doses, vaccination schedules, and outcome measurements have been reported (see http://DNAvaccine. com/ for a summary). One of the most thoroughly studied

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ARTICLE NAV systems has been vaccination with plasmid encoding the hepatitis B virus surface antigen (16 –18). To facilitate comparison with other NAV methods, the current studies employ this same HBV sAg coding plasmid to assess immune responses to NAV via skin electropermeabilization (EP) versus NAV alone. The large numbers of antigen-presenting cells (APC) (Langerhans cells and dermal dendritic cells) available for direct transfection provide an advantage for cutaneous NAV (19). Although APC transfection is not required for immune response to a transgene-encoded antigen (20), only professional antigen-presenting cells are able to induce cell-mediated immune responses due to expression of required costimulatory molecules (21). The few studies of direct plasmid delivery to skin suggest that the transfection efficiency is modest (22), as is the resulting NAV response (23). Biolistic transfection increases skin transfection efficiency, and this method does enable direct transfection of cutaneous dendritic cells with resulting humoral and cellular immune responses (24). In vivo DNA transfection may be enhanced by electric field treatment, a process termed electroporation or electropermeabilization. Under the influence of an electrical field, cellular membranes build up a transmembrane potential until the dielectric strength of the membrane is exceeded and a permeation event occurs. Continued or repeated application of a field after permeation facilitates additional beneficial alterations in membrane structure as well as nucleic acid transport. These membrane changes include increases in pore size required for transit of charged macromolecules (25). Recently, the term electropermeability has been used to describe this process (1, 26, 27), and methods involving in vivo electrical treatment of cells have been utilized to gain temporary access to the cytosol for many purposes (27). For example, EP has been used clinically to deliver chemotherapeutic agents to tumor cells including cutaneous malignancies (27, 28). The electrical parameters optimized for clinical electrochemotherapy are very similar to the protocol described in this report. The current studies are designed to test the hypothesis that in vivo skin EP will increase naked DNA transfection efficiency. Reasoning that NAV efficacy is limited by transfection efficiency, we also hypothesized that EP would enhance NAV responses relative to naked DNA alone. To test the first hypothesis, we have compared in vivo skin EP versus direct plasmid injection using various reporter genes in murine and porcine models. We then tested for the presence of transfected cells in lymph nodes draining the electropermeabilized skin. Finally, we compared the immune responses to a gene encoding hepatitis B virus surface antigen in electropermeabilized versus nonelectropermeabilized animals. The results indicate that EP increases transfection efficiency and broadens the range of transfected cell types and that these increases are associated with enhanced Th1-dominant immune responses to transgene-encoded antigen.

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MATERIALS

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METHODS

Plasmids and DNA preparation. The following CMV promoter-driven plasmids were employed in the experiments: pND2-lux, pEGFP-C1 (Clontech), pND ␤-gal (LacZ), all purified from transformed bacterial culture (Monster Prep, Bio 101, Carlsbad, CA). Plasmid encoding the hepatitis B surface antigen, pRc/CMV-HBs(S), was purified by Aldevron (Fargo, ND). Animals. Five- to seven-week BALB/c mice were purchased from Charles River (Wilmington, MA) and were anesthetized by an intraperitoneal injection of ketamine hydrochloride, xylazine hydrochloride, and acepromazine maleate before treatment. Four-week Yorkshire pigs were obtained from Tom Morris, Inc. (Reistertown, MD). Before treatment, pigs were anesthetized intramuscularly with ketamine and acepromazine. To harvest tissues for gene expression, mice were sacrificed by CO2 inhalation and pigs anesthetized as above followed by lethal pentobarbital injection. All animal work was approved by institutional animal use review board. Intradermal DNA injection and electroporation. The required concentration of each plasmid DNA (luciferase, pND2Lux, pEGFP, LacZ) was intradermally injected into pig and mouse. Sites on pigs included the ventral abdominal skin and limbs. The sites on mice were limited to the thicker skin just above the base of the tail. Total fluid volume in all experiments was 100 ␮l in injection-grade water. Either a caliper (plate) or a pin electrode consisting of two rows of seven 7-mm pins (1 ⫻ 5.4-mm gaps) was used to transfer the electric field to the injection site. The pin electrode penetrated about 2.5 mm into the animal skin. In all experiments, a sham treatment in which the electrode was placed on the injection site for 20 s without turning on electricity was performed to eliminate traumatic artifact. All EP experiments were performed using PulseAgile Electroporation equipment and software (CytoPulse Sciences, Hanover, MD). Detection of gene expression. Animals were euthanized 24 or 48 h after treatment and skin biopsies were obtained from the injection sites using an 8-mm disposable biopsy punch. Specific analyses was then carried out to determine gene expression levels for the different plasmid DNA used. Relative luciferase activity was determined (as described by the manufacturer) using the enhanced luciferase assay kit (Cat. No. 556866; Pharmingen, U.S.A.) and a Monolight luminometer 2010 (Analytical Luminescence Laboratories, San Diego, CA). Under ideal conditions with the enhanced luciferase assay kit, the specific activity of luciferase protein in this luminometer is 27,275 RLU/pg luciferase protein. In tissue lysates, measured luciferase specific activity is often substantially lower. Reagents for LacZ (␤-galactosidase) were obtained from Specialty Media (Phillipsburg, NJ) and used according to manufacturer’s recommendations. GFP skin sections were imaged fresh and/or fixed in 2% filtered paraformaldehyde solution for 1 h and soaked in 30% sucrose solution overnight (4°C) prior to sectioning. Each biopsy section (LacZ and EGFP) was individually embedded in OCT and frozen in liquid nitrogen. Serial cryostat sections (10 ␮m; Leica CM1900) were made and placed on polylysine slides (slides briefly soaked in 1:2 solution of polylysine and deionized water), sections were photographed, and images were analyzed for the respective reporter genes. Distribution of transgene mRNA expression was characterized by fixing murine skin biopsies in Parafix (1:20 tissue weight/solution). Sections were cut, and protease treated, and then hybridized with a 35S-labeled RNA antisense probe designed to recognize HBV sAg transcripts as previously described (29). For general histological analysis of skin tissues, biopsies were fixed in formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Detection and analysis of transgene expression in draining lymph nodes. One hundred microliters of methylene blue was injected s.q. into the skin at all of the transfection sites in the ventral aspect of the lower extremities of the pig 48 h after it received 10 separate plasmid injections of EGFP, pLUX, or control plasmid with EP in this area. The pig was euthanized after 30 min to allow for drainage through lymphatics. The draining lymph nodes were identified by their blue color and removed; generally three were identified per limb. They were then analyzed for appropriate transgene expression as described above. Quantitation of antibody response and immunoglobulin subclass analysis. Mice in groups of seven were immunized with i.d. injection of either pRc/CMV-HBs(S) as transgenic antigen or pND2-lux as a negative control MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy

ARTICLE followed by EP with pin electrode or sham treatment as above. As a positive control, five mice were inoculated with human recombinant hepatitis B vaccine (Recombivac-HB) with a dosing regimen of 0.05 ␮g in 50 ␮l per mouse. The human Recombivac-HB-vaccinated mice were on a different calendar schedule. This avoided cross contamination. Some groups were boosted using the same parameters as on day 0 treatment at week 3 or 9 or both. Antibody determinations were performed every 3 weeks. Antibody levels to hepatitis B surface antigen were determined by ELISA as previously described (30) using hepatitis B surface antigen purchased from Biodesign International. IgG subclass analysis was done using HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, and IgG2b (Southern Biotechnology Associates, Inc). IgG1:IgG2a ratios were determined for individual animals and compared as previously described to quantitate relative Th1 versus Th2 responses (31, 32).

RESULTS Effect of electropermeabilization on luciferase expression in murine and porcine skin. To optimize treatment conditions, a series of experimental and control intradermal injections of plasmid encoding firefly luciferase (pND2LUX) were performed on pigs and mice with and without EP as summarized in Fig. 1. Direct intradermal plasmid injection results in significant levels of transgene expression, and therefore all experiments were performed with an internal control of the same plasmid at the same dose without EP. The optimization protocols were designed to address the variables of electrical field strength, total number of pulses, electrode architecture, and DNA dose, with significant differences determined by t test. The experiments in mice (Fig. 1A) demonstrated that EP enhanced transgene expression under all tested treatment conditions (P ⬍ 0.01). For these experiments, an electrode consisting of an array of silver acupuncture needles was used to administer the electrical field pulses. In general, higher levels of transgene expression were observed with six versus two square-wave, 100-␮s pulses, except at the lowest field strength. Using the six-pulse protocol and varying electrical field strength, levels of transgene expression were observed to plateau at 1750 V/cm with a 16-fold increase relative to DNA alone (P ⬍ 0.02). Based on these observations, all subsequent in vivo EP treatments were performed using 1750 V/cm electrical fields administered as six square-wave 100-␮s pulses administered at 125-ms intervals. EP also enhanced transgene expression after injection into pig skin at varying doses of plasmid DNA (Fig. 1B). These studies were performed using the penetrating electrode described above. The difference in transgene expression relative to plasmid alone was most pronounced at the 50-␮g DNA dose (83-fold over free DNA, P ⬍ 0.005) and gradually tapered off as the DNA dose was increased from 100 ␮g (18.5-fold, P ⬍ 0.0003) to 200 ␮g (3.7-fold, P ⬍ 0.068) and 300 ␮g of plasmid (2.6-fold, P ⬍ 0.1). Based on these findings, a 100-␮g intradermal plasmid dose was selected for subsequent experiments. In general, in vivo EP treatments are performed with either flat plate or penetrating electrodes. Therefore, experiments were performed to compare the utility of these two electrode designs using the pig model (Fig. 1C). In this set of experiments, both electrodes provided a 26-fold enhancement of transgene expression relative to DNA MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy

FIG. 1. Effect of EP on quantitative luciferase expression in murine and porcine skin under varying conditions. Bars represent the means ⫾ standard deviations for the group. Units are in total relative light units in pg per punch biopsy homogenate. (A) Efficiency of transfection in mouse skin with/without EP using varying electrical field strength and number of applied pulses (100 ␮s per pulse at 125-ms intervals). (B) Efficiency of transfection in pig skin with/ without EP using varying DNA concentrations (6 pulses at 1750 V/cm, 100 ␮s per pulse at 125-ms intervals). (C) Efficiency of transfection by luciferase expression in pig skin with/without EP using different electrode types (6 pulses at 1750 V/cm, 100 ␮s per pulse at 125-ms intervals).

without EP (flat plate, P ⫽ 0.043; penetrating plate, P ⫽ 0.007). Similar results were also observed in the mouse model (data not shown). Because of ease of use and reduced variability in transgene expression, the penetrating electrode was selected for subsequent experiments. Effect of electropermeabilization on qualitative gene expression and distribution in murine and porcine skin. In addition to the quantitative increases in transgene expression

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ARTICLE pathologic tissue damage suggests that the procedure is well tolerated, consistent with the absence of behaviors suggesting discomfort from the treatment after emergence from anesthesia. Effect of electropermeabilization on transgene expression in lymph nodes draining electropermeabilized sites. The lower limbs of pigs were transfected with the LUX plasmid in multiple sites. The afferent draining lymph nodes were readily detected by methylene blue tracing. Forty-eight hours after transfection, luciferase activity in draining lymph nodes was low but significantly higher in nodes draining LUX-transfected skin as opposed to nodes from control skin transfected with LacZ (RLU mean 0.057 pg LUX-transfected skin vs 0 pg with LacZ-transfected skin with a P ⬍ 0.05 by Mann–Whitney test). Likewise, EGFPtransfected cells were noted in the draining lymph nodes from skin transfected with that transgene (Figs. 2E, 4⫻, and 2F, 40⫻) relative to control nodes (Fig. 2G, 40⫻). These experiments demonstrate that transfected cells are present in the lymph node and therefore may act to initiate an immune response by interacting with the lymphocytes therein.

FIG. 2. Effect of EP on qualitative gene expression and distribution in skin. Histopathologic analysis with EGFP and LacZ expression demonstrating a variety of morphologic phenotypes of transfected cells in electropermeabilized pig skin: (A) whole skin at 4⫻, (B) EGFP transfected adipocytes at 10⫻, (C) various EGFP-transfected dermal cells at 40⫻, (D) magnified views of EGFPtransfected dermal cells with fine cytoplasmic processes (consistent with dendritic cells) at 100⫻. EGFP expression in unfixed draining lymph nodes of EGFP at 4⫻ (E) and 40⫻ (F) versus (G) HepB (control) transfected limbs at 40⫻. In situ hybridization using labeled probe for hepatitis B mRNA in mouse skin transfected by electropermeabilization: (H) phosphor image analysis demonstrating distribution of injected DNA and transgene expression in EP-treated tissues, (I) collection of photoemulsion particles demonstrating mRNA expression in HBV sAg transfected cells. (All magnifications are original.)

noted in the luciferase studies, Fig. 2 demonstrates that a qualitative increase in transfection was observed with EP relative to naked DNA injection. For these studies, the distribution and frequency of transfection were assessed by histologic analysis of EGFP, LacZ (data not shown), or hepatitis B virus surface antigen transgene expression. EP of porcine skin using marker transgene EGFP demonstrated transfection of a wide variety of cell phenotypes which were concentrated in the dermis (Fig. 2A). Typical transfected cells included fibroblasts, endothelial cells, adipocytes (Figs. 2B and 2C), and mononuclear cells with elaborate cytoplasmic processes that may include dermal dendritic cells (Fig. 2D). Although these cells clearly have the morphology of dendritic cells, due to the lack of available reagents for swine surface cell markers, these cells were not characterized further. Lower transfection efficiency was observed with DNA alone, and the transfected cells were observed primarily in the epidermis. Hematoxylin and eosin staining of electroporated skin areas in both animal models showed little evidence of necrosis or inflammation (data not shown). The lack of

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Effect of electropermeabilization on immune responses to an antigen-encoding transgene. A plasmid encoding a wellcharacterized relevant antigen was used to determine if EP could augment immune response to the antigen. Murine skin was transfected with a hepatitis B antigen-encoding plasmid with a CMV promoter, pRc/CMV-Hbs(S), using EP. By 24 h, there was clear transcription of hepatitis B surface antigen mRNA as detected by in situ hybridization with a 35S-labeled antisense probe (Figs. 2H and 2I). The immune responses to hepatitis B in mice immunized with the plasmid encoding hepatitis B surface antigen delivered with and without EP were compared by ELISA using whole IgG (Fig. 3). Mice immunized with the hepatitis B DNA alone demonstrated no measurable humoral response to hepatitis B sAg despite repeated boost-

FIG. 3. Antibody responses to hepatitis B surface antigen immune responses over time in mice transfected with/without EP and with/without boosting. Bars represent mean IgG titers for different groups. Serum for each animal was run separately (not pooled) and mean titers plus standard errors of the mean were calculated. MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy

ARTICLE TABLE 1

Immunization treatment group Recomgivac sq (0.05 ␮g/mouse in 50 ␮l), no boost HBV DNA alone, boost at 3 and 9 weeks HBV DNA, EP 1500 V/cm, 6 pulse, boost at 3 and 9 weeks HBV DNA, EP 1750 V/cm, 6 pulse, boost at 3 and 9 weeks

IgG1:IgG2a ratio ⬎ 1.0 over final total N per group

IgG1:IgG2a ratio ⱕ 1.0 over final total N per group

126, 128, 8, 1280, 80

5/5

0/5

0% Th1 100% Th2

0, 0, 0, 0, 0, 0

—, —, —, —, —, —

Indeterminate

Indeterminate

Indeterminate

80, 640, 320, 10240, 2560, 0, 40 0, 1260, 320, 0, 1260, 0, 80

0.0125, 0.5, 0.5, 0.5, 0.0003, 20, 64

2/7

5/7

71% Th1 dominant 29% Th2 dominant

—, 0.25, 0.5, 20, 8.1, —, 1

2/5 responders

3/5 responders

60% Th1 dominant 40% Th2 dominant

IgG titers for all mice (week 12)a

IgG1 titers for all mice (week 12)a

IgG2A titers for all mice (week 12)a

40480, 2560, 160, 1280, 80 0, 0, 0, 0, 0, 0

40480, 2560, 160, 1280, 80 0, 0, 0, 0, 0, 0

320, 20, 0, 0, 0

80, 640, 320, 10240, 2560, 20, 40 0, 1260, 320, 20, 10240, 0, 80

0, 320, 160, 5120, 0, 20, 2560 0, 320, 160, 20, 10240, 0, 80

IgG1:IgG2a ratios in individual animals

Th1 versus Th2 response

Note. Data for immunization groups are tabulated. Sera were obtained 12 weeks after primary immunizations. All IgG, IgG1, and IgG2a serum titers are shown. Actual ratios for individual animals with positive IgG1 or IgG2a titers are listed. Number of animals with high (⬎1.0) versus low (ⱕ1.0) IgG1:IgG2a ratios over the total final N per group are compared for each immunization treatment. High IgG1:IgG2a ratio (ⱕ1.0) suggests a Th2-dominated response and low ratio (ⱕ1.0) suggests a Th1-dominated response. a All units in column represent 1:n dilutions.

ing, while animals receiving hepatitis B DNA with EP developed a sustained humoral immune response by week 9. Animals given a single primary immunization developed titers somewhat less than those given boosts at 3 weeks, suggesting that primary immunization plus boosting enhances the sustained immune response (data not shown). The total IgG response to 1500 V/cm was equivalent to that of 1750 V/cm (Fig. 3). Quantitative IgG subclass analysis revealed that both IgG1 and IgG2a antibodies were produced, consistent with a mixed Th1/Th2 response (Table 1). The majority of responding animals had low IgG1:IgG2a ratios, suggesting that a Th1 response was dominant. This is in contrast to animals immunized with Recombivac, who developed a pure IgG1 (Th2) response pattern. This difference is statistically significant by Fischer’s exact test at P ⫽ 0.02. The humoral responses engendered by 1500 V/cm EP appeared equivalent to those observed in 1750V/cm EP-treated animals. All animals inoculated with human recombinant hepatitis B vaccine had a Th2 profile (Table 1). Treatment groups immunized with luciferase DNA and HBV DNA ⫹ EP (no boost) are not included in this table.

DISCUSSION In this report, we demonstrate that the in vivo application of pulsed electric fields significantly increases intradermally injected transgene expression. The procedure is easy to perform and well tolerated both clinically and histopathologically. In contrast to the predominantly epidermal pattern of expression observed after intradermal injection of naked DNA (22), a pattern of dermal and subdermal transfection was observed with skin EP. Transfected cell types included numerous mononuclear cells MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy

with dendritic cell morphology, as well as large numbers of adipocytes. Transgene expression was observed in lymph nodes draining electroporated tissues, although the studies do not distinguish between the migration of transfected cells from skin to nodes and the direct uptake and transfection of resident lymph node cells by the injected DNA. To test the hypothesis that increased transfection efficiency would translate into increased nucleic acid vaccination efficacy, mice were vaccinated with a HBV surface antigen-encoding plasmid. Analysis of resulting humoral immune response patterns demonstrated that EP treatment strongly enhanced NAV relative to immunization with DNA alone. This response was mixed but Th1 dominant compared to immunization with protein alone. An early experiment by Titomirov and Sukharev (33) demonstrated that DNA EP into newborn mouse skin yielded stably transfected fibroblasts, and others have demonstrated that the topical application of naked DNA followed by EP was superior to DNA alone (34 –36). In these experiments, it was hypothesized that EP allowed passage of the DNA through the physical barrier of the stratum corneum by burning microconduits. In our experiments, the stratum corneum was bypassed by direct intradermal injection, and the addition of pulsed electric fields allowed for the transfection of a wide variety of dermal cell types. This pattern of transgene expression has not been previously described. The current work demonstrates that monocytes with elaborate dendritic processes are readily transfected by EP, perhaps due to the inverse relationship between local membrane radius and poration efficiency (37). Alternatively, preferential transfection of such cells may reflect the wider effective cell width due to the processes. The

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ARTICLE preferential EP-mediated transfection of cells with dendritic morphology could provide a distinct advantage for NAV due to the central role of such cells in the stimulation of both primary and secondary immune responses. Dermal adipocytes also appear to be particularly susceptible to transfection via electroporation. From the perspective of electroporation, adipocytes may be modeled as large cells with a thin rim of cytoplasm surrounding a large, nonconducting vacuole. Both overall cell size and the high surface-to-electrolyte volume ratio may enhance EP-mediated transfection in part by reducing the effective threshold field strength required for EP. We found that EP augments the magnitude of transfection which may allow for lower i.d. DNA doses to be used for nucleic acid vaccination. Surprisingly, we noted no effect of electrode design (penetrating pin versus caliper plate) in these experiments. We also conclude that multiple pulses (greater than two) enhance skin transfection and that the benefit of an increased field strength appears to plateau beyond 1750 V/cm. With the possible exception of bone and stratum corneum, tissues have interstitial fluid that acts as both a resistor and a conductor. In such an environment, heat is related to the square of the voltage, and therefore increasing the applied voltage may overheat the cells. Therefore, we hypothesize that increasing field strength beyond 1750 V/cm results in currentmediated damage that may decrease transfection efficiency due to necrosis and/or inflammation (38). The main thrust of this work was to determine if intradermal electropermeabilization was superior to naked DNA for nucleic acid vaccination. Key findings supporting EP-mediated NAV include the superior quantitative transfection of skin cells with in vivo EP, the direct transfection of cells morphologically consistent with dendritic cells, and expression of both luciferase and EGFP marker genes in afferent draining lymph nodes. Dendritic cell migration after biolistic transfection with gold beads has also been described (24). It is known that activated dendritic cells migrate to afferent nodes within hours (39). The presence of transfected cells in the lymph nodes suggests that skin dendritic cells were directly transfected by the marker genes in these experiments, became activated, and migrated to draining lymph nodes. The direct transfection of antigen-presenting cells may be used to induce cellular immune responses and may break immunologic tolerance (18). The triggers for the immune activation and migration remain unclear, but may be due to the process of transfection itself in which foreign DNA is introduced into the cytoplasm (40). CpG immunostimulatory motifs in the plasmid DNA may play an added role (41). Likewise, there may be a nonspecific effect of electricity or ballistic trauma in the area that is too subtle to be detected on histopathologic analysis, associated with release of “danger signals” and activation of local dendritic cells (42). Our final set of experiments examined the humoral immune response to an injected polynucleotide vaccine with and without EP. Using in situ hybridization to detect transcribed mRNA, it was demonstrated that naked DNA

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injection with EP resulted in expression of the transgene encoding hepatitis B surface antigen. In these studies, mice that received the hepatitis B plasmid without EP developed no measurable antibody response to the transgene despite a primary injection and two boosts. However, after reviewing the literature, this result is not surprising. pRc/CMV-HBs(S) is a “first-generation” plasmid that is not nearly as immunogenic as many of its current counterparts (43– 47). The i.d. route is notorious for its lower antibody responses compared to intramuscular routes of vaccination in mice (45– 47). Furthermore, transfection efficiency is much lower and results vary when using an intradermal route compared to gold-gun or intramuscular vaccination (20). In contrast, all groups of animals that received EP in concert with intradermal injection mounted an increased immune response despite the potential limitation described above. In EP-treated animals, boosting was of measurable benefit although animals that received only the primary vaccination still mounted a response by 9 weeks. The IgG antibodies in these animals were a mix of subclasses IgG1 and IgG2a, suggesting a mixed Th1 and Th2 response. A low IgG1: IgG2a ratio was observed in the majority of animals in all EP immunization groups, suggesting Th1 dominance (49). As with the total IgG response, the 1500 and 1750 V/cm groups gave similar Th1-dominant subclass ratios. A Th1 response may be a desirable advantage of this technique, since cellular immune mechanisms are more important in dealing with chronic infections and neoplasms, disorders to which vaccine therapy may be targeted (50). The recombinant protein-inoculated group elicited a 100% Th2-biased response to the vaccine (Table 1). Thus, the NAV-EP groups show that 66% of mice have a Th1 profile, compared to 0% of the recombinant protein-inoculated group (P ⫽ 0.02). Future directions for this work include characterizing cellular responses after DNA vaccination. However, this first report clearly demonstrates that intradermal vaccination combined with electroporation significantly increases immune responses with a mixed Th1:Th2 profile with Th1 predominating. In summary, we have shown that in vivo EP-mediated skin transfection is easy, safe, and more effective than the injection of naked DNA alone. A wide variety of cell types are transfected, and transfected cells can be found in the draining afferent lymph nodes, suggesting migration of directly transfected dendritic cells. Consistent with these findings, we noted that only mice immunized by pRc/ CMV-Hbs(S) plasmid DNA plus electropermeabilization mounted a measurable immune response against the antigen and that this response was of a mixed Th1 and Th2 pattern, favoring the former. This was in distinct contrast to protein immunization, which yielded an exclusive Th2 response. These studies demonstrate that skin electropermeabilization for gene transfer may have broad applicability for nucleic acid vaccination. ACKNOWLEDGMENTS The views of these authors do not purport to be those of the United States government or the Department of Defense. CytoPulse and the University of MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy

ARTICLE Maryland, Baltimore, have a collaboration agreement under which CytoPulse’s equipment is used by Robert Malone, M.D., in scientific research and development, principally in the areas of delivering DNA using pulsed electric fields. These studies were supported by NIH-K02AI01370 (R.W.M.); NIHR01RR12307 (R.W.M.); and developmental funds provided by the University of Maryland Medical System (R.W.M).

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