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Enhancement of DNA vaccine potency by electroporation in vivo
Journal of Biotechnology 83 (2000) 147 – 152 www.elsevier.com/locate/jbiotec
Enhancement of DNA vaccine potency by electroporation in vivo Mark Selby a, Cheryl Goldbeck a, Terry Pertile b, Robert Walsh b, Jeffrey Ulmer a,* a
Vaccines Research, Chiron Corporation, 4560 Horton Street, Emery6ille, CA 94608, USA b − e Med Corporation, St Paul, MN 55112, USA
Received 6 September 1999; received in revised form 1 December 1999; accepted 9 December 1999
Abstract The potential of electric current-mediated delivery technology to enhance DNA delivery and DNA vaccine potency was evaluated. Higher levels of reporter gene expression were observed in muscle cells of mice inoculated with luciferase or b-galactosidase DNA followed by the application of electrical current, compared with DNA injected with no current. Similarly, substantially higher levels of immune responses (up to 20-fold) were demonstrated in mice vaccinated with HIV gag DNA and electric current. These enhanced responses were observed after one or two inoculations, and were maintained for at least 12 weeks. Therefore, the present studies demonstrate the utility of electroporation for enhancement of DNA vaccine potency in animals. © 2000 Elsevier Science B.V. All rights reserved. Keywords: DNA; Electroporation; Vaccine potency
1. Introduction The efficacy of DNA vaccines in preclinical animal models has been well documented (for review see Donnelly et al., 1997). However, the magnitude of immune responses induced in primates is generally lower than that in small animals, and the amount of DNA required for
* Corresponding author. Tel.: +1-510-9235140; fax: +1510-6580329. E-mail address: jeffrey – [email protected] (J. Ulmer).
effective immunization of primates is much higher (mg versus mg) (for example see Gramzinski et al., 1998; Kent et al., 1998; Richmond et al., 1998). In addition, several phase I human clinical studies have been conducted with little or no immune responses reported (Calarota et al., 1998; MacGregor et al., 1998). In one case, however, cytotoxic T lymphocytes were induced in human volunteers by a malaria DNA vaccine, but no antibodies were detected (Wang et al., 1998). Therefore, the potency of DNA vaccines must be increased to enable this technology for successful human application.
0168-1656/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 0 ) 0 0 3 0 8 - 4
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M. Selby et al. / Journal of Biotechnology 83 (2000) 147–152
The use of electric current has facilitated gene delivery in vitro and in vivo. Transient discontinuities in the plasma membranes of cells can be induced by short pulses of high-voltage electric current. This allows substances, such as plasmid DNA, to passively enter cells directly into the cytoplasm, thereby avoiding the indirect and potentially inefficient route of endocytosis. As a consequence, more DNA is delivered inside cells and a greater degree of transfection occurs. This process, termed electroporation, is widely used for facilitation of transfection of cells in vitro. Recently, the use of electric current to mediate transfer of genes in vivo has been reported. Successful transfer of genes has been accomplished for cells of the skin (Titomirov et al., 1991; Nomura et al., 1996), liver (Heller et al., 1996; Suzuki et al., 1998), tumors (Nishi et al., 1996, 1997; Rols et al., 1998), oviduct (Ochiai et al., 1998), and muscle (Aihara and Miyazaki, 1998). In most cases, protein expression was demonstrated, and in some cases biological effects were noted, such as regression of tumors or increased hematocrit after inoculation of erythropoietin DNA (Rizzuto et al., 1999). In one case, induction of an immune response was detected in mice after electroporation in vivo with DNA encoding a fusion protein containing a CTL epitope from influenza nucleoprotein (Nomura et al., 1996). In this case, though, no comparison was made to inoculation of DNA without current. A technology related to electroporation, termed iontophoresis, involves the application of an electric field to facilitate movement of charged molecules in tissue and across biological membranes. Iontophoresis, which involves lower levels of constant current rather than the higher levels of constant voltage required for electroporation, has been widely used for transdermal delivery of drugs and oligonucleotides. However, there have been no published reports of iontophoretic delivery of plasmid DNA. The present study was conducted to evaluate the potential of iontophoresis and electroporation to facilitate DNA vaccine delivery in vivo leading to enhancement of immune responses.
2. Material and methods
2.1. Bacterial strain and plasmid preparation Escherichia coli strain HB101 were transformed with the plasmids pCMV HIV p55 or pCMVKM LUC encoding firefly luciferase reporter gene (LUC). The plasmids were purified using a Qiagen Endofree Plasmid Giga kit (Qiagen, Hilden, Germany) and resuspended in 0.9% sodium chloride (Abbott Laboratories, North Chicago, IL). The concentrations were analyzed by measuring absorbance at 260 nm.
2.2. Immunization procedure Female 6–8-week-old CB6F1 mice (Charles River) were anesthetized using four parts ketamine HCl, 100 mg ml − 1 stock solution (Fort Dodge Animal Health, Fort Dodge, Iowa), one part xylazine, 20 mg ml − 1 (Lloyd Labs, Shenandoah, Iowa). The mice received 1 ml mg − 1 of body weight intramuscularly in the posterior thigh. The tibialis anterior (TA) muscle was shaved and the animals were injected with 10 mg of plasmid in a volume of 50 ml. To control needle depth, a 0.3 cc insulin syringe was covered with polyethylene tubing (i.d. 0.38) to expose only the bevel. In some instances, electric current was applied to the injected muscles as follows. For constant current deliveries (iontophoresis), plasmid DNA in 5% dextrose was injected into the right tibialis anterior muscle using a single needle delivery probe, which has a functional length of 3 mm. Following plasmid injection, the plasmid delivery needle was attached to the negative lead from the controller and a needle electrode placed in the contralateral leg was attached to the positive lead. Constant current pulses of 5 mA in amplitude, 10 ms in width, were given at a frequency of 1 Hz for 1 min. For constant voltage deliveries (electroporation), plasmid DNA in PBS was injected into the right tibialis anterior muscle as previously described. Electrical energy delivery was performed through a bipolar needle probe that was placed over the site of plasmid injection. The probe needles had a separation distance of 0.4 cm
M. Selby et al. / Journal of Biotechnology 83 (2000) 147–152
and a needle length of 0.3 cm. The probe was connected to a constant voltage power supply and five constant voltage pulses, 50 ms in width, either 100 or 200 V cm − 1, were applied in one orientation, the probe was rotated 90° and five additional pulses were applied.
2.3. Measurement of luciferase acti6ity Mice were sacrificed 1 – 7 days post vaccination, and TA muscles were collected and flash frozen in liquid nitrogen. The frozen tissue was homogenized with a mortar and pestle (on dry ice), lysed with 0.5 ml 1X reagent lysis buffer (Promega, Madison, WI), and vortexed for 15 min at room temperature. The samples were subjected to three freeze thaws and centrifuged for 10 min at 10 000×g. Supernatants were collected and stored at − 80°C until assayed. The ML3000 microplate luminometer (Dynex Technologies, Chantilly, VA) measured the luciferase activity by automatically dispensing 100 ml of luciferase assay reagent (Promega, Madison, WI) into wells containing 20 ml of supernatant, and measuring the relative light units (RLU). The setting for the luminometer were the following, Mode: enhanced flash, Gain: medium, Delay time: 1 s, Integrate time: 5 s, calibrate each run. Sample values were extrapolated from a standard curve prepared from QuantiLum® Recombinant Luciferase (Promega, Madison, WI). Results are expressed as ng luciferase per mg muscle protein, with protein determination by BCA Protein Assay Reagent (Pierce).
2.4. Measurement of antibody responses Serum samples were collected at selected intervals and anti-gag antibodies were measured by a chemi-luminescent ELISA assay. MicroLite 2, 96well flat bottom plates (Dynex Technologies, Chantilly, VA) were coated with HIV p24 protein at 5 mg ml − 1 in 10 mM tris pH 7.5, 50 ml per well and incubated at 4°C overnight. The plates were washed three times with wash buffer (1X AquaLite® Wash Buffer (SeaLite Sciences, Inc., Bogart, GA) containing 0.3% Tween 20 (Sigma, St Louis, MO)), and blocked at 37°C for 1 h with
149
150 ml per well blocking buffer (1X Streptavidin AquaLite® Assay buffer (SeaLite Sciences, Inc., Bogart, GA) containing 5% goat serum). The plates were washed 3X and the test sera were diluted 1/300 or 1/9000 followed by serial threefold dilutions in the blocking buffer. A volume of 50 ml of each dilution was added per well and the plates were incubated at 37°C for 1 h. The plates were washed six times and incubated for 1 h at 37°C with 50 ml per well of Goat anti-mouse IgG-Biotin (Sigma St. Louis, MO), diluted 1/1000 in block buffer. After washing six times, the plates were incubated at 37°C for 1 h with Streptavidin– Aqualite® (SeaLite Sciences, Inc., Bogart, GA), diluted 1/500 in wash buffer, 50 ml per well. The plates were washed six times and stored in wash buffer until reactivity was measured on the luminometer (MLX, Dynex Technologies, Chantilly, VA). Setting for the luminometer—mode: Integrate Flash, Gain: High, Data: Table, Delay window: 0.00 s, Integrate window: 3.00 s, Before peak: 0.10 s, After peak: 2.00 s, calibrate on each well. The plates were tapped dry and put into the luminometer. Fifty microliters of 1X AquaLite® Trigger Buffer (SeaLite Sciences, Inc., Bogart, GA) were automatically dispensed per well and the relative light units (RLU) were measured. Endpoint titers were calculated as the inverse of the dilution that yields an RLU equal to the background plus five times the standard deviation.
3. Results and discussion Previous reports have demonstrated that application of electric current after injection of plasmid DNA has resulted in increased expression of the encoded proteins in the injected tissues (for example see Mathiesen, 1999; Mir et al., 1999). In this study, we used DNA plasmids encoding the reporter genes luciferase and b-galactosidase to measure transfection of muscles cells in vivo. At 4 days after a single inoculation of DNA, luciferase expression was higher in muscles treated with electric current compared to untreated muscles (Table 1). This was true for both iontophoresis (4.6-fold) and electroporation (7.3-fold). Simi-
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larly, the number of muscle fibers detectably transfected after inoculation of b-galactosidase DNA was substantially increased by iontophoresis and electroporation compared to untreated muscles, as judged by b-galactosidase staining of muscle tissue sections (data not shown). In addition, as noted previously (Mir et al., 1998), application of electric current appears to decrease the variability of reporter gene expression in muscle cells. Therefore, application of electric current facilitates delivery of DNA to muscle cells in situ and, hence, may be able to overcome some of the barriers to efficient transfection. To assess the effect of electric current on the immunogenicity of a DNA vaccine, mice received a single immunization with HIV gag DNA (10 mg) with or without electric current, and sera from mice were analyzed at 2, 4, 8 and 12 weeks after inoculation. At all time points tested, both iontophoresis and electroporation enhanced antibody titers in mice compared to those receiving no further treatment, with enhancement ranging from 8- to 20-fold (Fig. 1). As with luciferase expression levels, in general, electroporation conditions ap-
peared slightly superior to iontophoresis for enhancement of antibody responses. In a separate study, groups of mice were immunized twice with HIV gag DNA with a 6 week interval and antibody responses were measured after each immunization. Antibody titers were elevated in all groups after the booster injection, but the tenfold enhancement in titers observed in mice receiving electric current was maintained even after the boost (Fig. 2). It is not yet known how iontophoresis or electroporation increased DNA vaccine potency, but one reasonable possibility is that the distribution of DNA within the injected tissue and/or uptake of DNA by cells was facilitated by the electric current leading to increased transfection. The ensuing increase in mass of antigen expressed by cells likely played a role in the elevated immune responses. However, it is also possible that infiltration of inflammatory cells in response to the electric current could have taken place resulting in an ‘adjuvant’ effect on the antigen produced. Further work will be required to fully evaluate the mode of action of this delivery technology.
Table 1 Enhancement of gene expression in muscle cells in vivoa Treatment Luc DNA (no current) (10 mg)
Luc DNA (ionto) (10 mg)
Luc DNA (electro) (10 mg)
Luc activity 6.76 0.74 0.44 9.11 1.92 26.63 10.54 23.46 5.51 20.61 18.5 35.02 39.02 33.22 13.06
Mean (S.D.)
Fold increase
3.794 (3.91)
1.00
17.35 (8.95)
4.57
27.764 (11.30)
7.32
a Groups of five CB6 F1 mice were inoculated with 25 mg of luciferase (Luc) DNA in the TA muscle of one leg. One group of mice was not further treated, one group was treated with iontophoresis (ionto) and another with electroporation (electro). At 4 days after inoculation, the muscles were collected and luciferase activity was measured and expressed as ng luciferase activity per mg muscle protein. Numbers in brackets indicate standard deviation of the mean (S.D.).
M. Selby et al. / Journal of Biotechnology 83 (2000) 147–152
151
Fig. 1. Enhancement of antibody responses in mice. Groups of four to six CB6 F1 mice were inoculated a single time with 10 mg of HIV gag DNA in the TA muscle of one leg. One group of mice was not further treated, one group was treated with iontophoresis and another with electroporation. At the indicated intervals, sera were collected and analyzed for antibody responses. Data are plotted as geometric mean ELISA titer and error bars indicate S.E.M.
In summary, we have demonstrated that DNA vaccine potency can be increased by application of electric current, likely as a consequence of increased delivery of DNA to and into cells. These results indicate that a significant limitation to efficient transfection of cells in vivo by naked DNA vaccines is distribution within tissue and/or uptake of DNA by cells. It is not yet known if this limitation contributes to the lack of efficacy of DNA vaccines in larger animals, such as primates. If so, iontophoresis, electroporation and other means designed to facilitate delivery of DNA hold promise as second generation DNA vaccine technologies.
References
Fig. 2. Effect of boosting on antibody responses in mice. Groups of six CB6 F1 mice were inoculated with 10 mg of HIV gag DNA in the TA muscle of one leg at 3 and 6 weeks. One group of mice was not further treated (open bars), one group was treated with iontophoresis (solid bars) and another with electroporation (shaded bars). Sera were collected at 3 weeks after each immunization and analyzed for antibody responses. Data are plotted as geometric mean ELISA titer and error bars indicate S.E.M.
Aihara, H., Miyazaki, J., 1998. Gene transfer into muscle by electroporation in vivo. Nature. Biotechnol. 16, 867 – 870. Calarota, S., Bratt, G., Nordlund, S., et al., 1998. Cellular cytotoxic response induced by DNA vaccination in HIV-1infected patients. Lancet 351, 1320 – 1325. Donnelly, J., Ulmer, J., Shiver, J., Liu, M., 1997. DNA Vaccines. Ann. Rev. Immunol. 15, 617 – 648. Gramzinski, R., Millan, C., Obaldia, N., Hoffman, S., Davis, H., 1998. Immune response to a hepatitis B DNA vaccine in Aotus monkeys: a comparison of vaccine formulation, route, and method of administration. Mol. Med. 4, 109 – 118.
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Heller, R., Jaroszeski, M., Atkin, A., et al., 1996. In vivo gene electroinjection and expression in rat liver. FEBS Lett. 389, 225 – 228. Kent, S., Zhao, A., Best, S., et al., 1998. Enhanced T-cell immunogenicity and protective efficacy of a human immunodeficiency virus type 1 vaccine regimen consisting of consecutive priming with DNA and boosting with recombinant fowlpox virus. J. Virol. 72, 10180–10188. MacGregor, R., Boyer, J., Ugen, K., et al., 1998. First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response. J. Infect. Dis. 178, 92–100. Mathiesen, I., 1999. Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther. 6, 508–514. Mir, L., Bureau, M., Rangara, R., Schwartz, B., Scherman, D., 1998. Long-term, high level in vivo gene expression after electric pulse-mediated gene transfer into skeletal muscle. CR. Acad. Sci. III 321, 893–899. Mir, L., Bureau, M., Gehl, J., et al., 1999. High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc. Natl. Acad. Sci. USA 96, 4262–4267. Nishi, T, Yoshizato, K., Yamashiro, S., et al., 1996. High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation. Cancer Res. 56, 1050 – 1055. Nishi, T., Dev, S., Yoshizato, K., Kuratsu, J., Ushio, Y., 1997. Treatment of cancer using pulsed electric field in combina-
tion with chemotherapeutic agents or genes. Hum. Cell 10, 81 – 86. Nomura, M., Nakata, Y., Inoue, T., et al., 1996. In vivo induction of cytotoxic T lymphocytes specific for a single epitope introduced into an unrelated molecule. J. Immunol. Meth. 193, 41 – 49. Richmond, J., Lu, S., Santoro, J., et al., 1998. Studies of the neutralizing activity and avidity of anti-human immunodeficiency virus type 1 env antibody elicited by DNA priming and protein boosting. J. Virol. 72, 9092 – 9100. Rizzuto, G., Cappelletti, M., Maione, D., et al., 1999. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc. Natl. Acad. Sci. USA 96, 6417 – 6422. Rols, M.P., Delteil, C., Golzio, M., et al., 1998. In vivo electrically mediated protein and gene transfer in murine melanoma. Nature Biotechnol. 16, 168 – 171. Suzuki, T., Shin, B.-C., Fujikura, K., Matsuzaki, T., Takata, K., 1998. Direct gene transfer into rat liver cells by in vivo electroporation. FEBS Lett. 425, 436 – 440. Titomirov, A., Sukharev, S., Kistanova, E., 1991. In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim. Biophys. Acta 1088, 131 – 134. Wang, R., Doolan, D.L., Le, T.P., et al., 1998. Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science 282, 476 – 480.
.
Enhancement of DNA vaccine potency by electroporation in vivo Mark Selby a, Cheryl Goldbeck a, Terry Pertile b, Robert Walsh b, Jeffrey Ulmer a,* a
Vaccines Research, Chiron Corporation, 4560 Horton Street, Emery6ille, CA 94608, USA b − e Med Corporation, St Paul, MN 55112, USA
Received 6 September 1999; received in revised form 1 December 1999; accepted 9 December 1999
Abstract The potential of electric current-mediated delivery technology to enhance DNA delivery and DNA vaccine potency was evaluated. Higher levels of reporter gene expression were observed in muscle cells of mice inoculated with luciferase or b-galactosidase DNA followed by the application of electrical current, compared with DNA injected with no current. Similarly, substantially higher levels of immune responses (up to 20-fold) were demonstrated in mice vaccinated with HIV gag DNA and electric current. These enhanced responses were observed after one or two inoculations, and were maintained for at least 12 weeks. Therefore, the present studies demonstrate the utility of electroporation for enhancement of DNA vaccine potency in animals. © 2000 Elsevier Science B.V. All rights reserved. Keywords: DNA; Electroporation; Vaccine potency
1. Introduction The efficacy of DNA vaccines in preclinical animal models has been well documented (for review see Donnelly et al., 1997). However, the magnitude of immune responses induced in primates is generally lower than that in small animals, and the amount of DNA required for
* Corresponding author. Tel.: +1-510-9235140; fax: +1510-6580329. E-mail address: jeffrey – [email protected] (J. Ulmer).
effective immunization of primates is much higher (mg versus mg) (for example see Gramzinski et al., 1998; Kent et al., 1998; Richmond et al., 1998). In addition, several phase I human clinical studies have been conducted with little or no immune responses reported (Calarota et al., 1998; MacGregor et al., 1998). In one case, however, cytotoxic T lymphocytes were induced in human volunteers by a malaria DNA vaccine, but no antibodies were detected (Wang et al., 1998). Therefore, the potency of DNA vaccines must be increased to enable this technology for successful human application.
0168-1656/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 0 ) 0 0 3 0 8 - 4
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M. Selby et al. / Journal of Biotechnology 83 (2000) 147–152
The use of electric current has facilitated gene delivery in vitro and in vivo. Transient discontinuities in the plasma membranes of cells can be induced by short pulses of high-voltage electric current. This allows substances, such as plasmid DNA, to passively enter cells directly into the cytoplasm, thereby avoiding the indirect and potentially inefficient route of endocytosis. As a consequence, more DNA is delivered inside cells and a greater degree of transfection occurs. This process, termed electroporation, is widely used for facilitation of transfection of cells in vitro. Recently, the use of electric current to mediate transfer of genes in vivo has been reported. Successful transfer of genes has been accomplished for cells of the skin (Titomirov et al., 1991; Nomura et al., 1996), liver (Heller et al., 1996; Suzuki et al., 1998), tumors (Nishi et al., 1996, 1997; Rols et al., 1998), oviduct (Ochiai et al., 1998), and muscle (Aihara and Miyazaki, 1998). In most cases, protein expression was demonstrated, and in some cases biological effects were noted, such as regression of tumors or increased hematocrit after inoculation of erythropoietin DNA (Rizzuto et al., 1999). In one case, induction of an immune response was detected in mice after electroporation in vivo with DNA encoding a fusion protein containing a CTL epitope from influenza nucleoprotein (Nomura et al., 1996). In this case, though, no comparison was made to inoculation of DNA without current. A technology related to electroporation, termed iontophoresis, involves the application of an electric field to facilitate movement of charged molecules in tissue and across biological membranes. Iontophoresis, which involves lower levels of constant current rather than the higher levels of constant voltage required for electroporation, has been widely used for transdermal delivery of drugs and oligonucleotides. However, there have been no published reports of iontophoretic delivery of plasmid DNA. The present study was conducted to evaluate the potential of iontophoresis and electroporation to facilitate DNA vaccine delivery in vivo leading to enhancement of immune responses.
2. Material and methods
2.1. Bacterial strain and plasmid preparation Escherichia coli strain HB101 were transformed with the plasmids pCMV HIV p55 or pCMVKM LUC encoding firefly luciferase reporter gene (LUC). The plasmids were purified using a Qiagen Endofree Plasmid Giga kit (Qiagen, Hilden, Germany) and resuspended in 0.9% sodium chloride (Abbott Laboratories, North Chicago, IL). The concentrations were analyzed by measuring absorbance at 260 nm.
2.2. Immunization procedure Female 6–8-week-old CB6F1 mice (Charles River) were anesthetized using four parts ketamine HCl, 100 mg ml − 1 stock solution (Fort Dodge Animal Health, Fort Dodge, Iowa), one part xylazine, 20 mg ml − 1 (Lloyd Labs, Shenandoah, Iowa). The mice received 1 ml mg − 1 of body weight intramuscularly in the posterior thigh. The tibialis anterior (TA) muscle was shaved and the animals were injected with 10 mg of plasmid in a volume of 50 ml. To control needle depth, a 0.3 cc insulin syringe was covered with polyethylene tubing (i.d. 0.38) to expose only the bevel. In some instances, electric current was applied to the injected muscles as follows. For constant current deliveries (iontophoresis), plasmid DNA in 5% dextrose was injected into the right tibialis anterior muscle using a single needle delivery probe, which has a functional length of 3 mm. Following plasmid injection, the plasmid delivery needle was attached to the negative lead from the controller and a needle electrode placed in the contralateral leg was attached to the positive lead. Constant current pulses of 5 mA in amplitude, 10 ms in width, were given at a frequency of 1 Hz for 1 min. For constant voltage deliveries (electroporation), plasmid DNA in PBS was injected into the right tibialis anterior muscle as previously described. Electrical energy delivery was performed through a bipolar needle probe that was placed over the site of plasmid injection. The probe needles had a separation distance of 0.4 cm
M. Selby et al. / Journal of Biotechnology 83 (2000) 147–152
and a needle length of 0.3 cm. The probe was connected to a constant voltage power supply and five constant voltage pulses, 50 ms in width, either 100 or 200 V cm − 1, were applied in one orientation, the probe was rotated 90° and five additional pulses were applied.
2.3. Measurement of luciferase acti6ity Mice were sacrificed 1 – 7 days post vaccination, and TA muscles were collected and flash frozen in liquid nitrogen. The frozen tissue was homogenized with a mortar and pestle (on dry ice), lysed with 0.5 ml 1X reagent lysis buffer (Promega, Madison, WI), and vortexed for 15 min at room temperature. The samples were subjected to three freeze thaws and centrifuged for 10 min at 10 000×g. Supernatants were collected and stored at − 80°C until assayed. The ML3000 microplate luminometer (Dynex Technologies, Chantilly, VA) measured the luciferase activity by automatically dispensing 100 ml of luciferase assay reagent (Promega, Madison, WI) into wells containing 20 ml of supernatant, and measuring the relative light units (RLU). The setting for the luminometer were the following, Mode: enhanced flash, Gain: medium, Delay time: 1 s, Integrate time: 5 s, calibrate each run. Sample values were extrapolated from a standard curve prepared from QuantiLum® Recombinant Luciferase (Promega, Madison, WI). Results are expressed as ng luciferase per mg muscle protein, with protein determination by BCA Protein Assay Reagent (Pierce).
2.4. Measurement of antibody responses Serum samples were collected at selected intervals and anti-gag antibodies were measured by a chemi-luminescent ELISA assay. MicroLite 2, 96well flat bottom plates (Dynex Technologies, Chantilly, VA) were coated with HIV p24 protein at 5 mg ml − 1 in 10 mM tris pH 7.5, 50 ml per well and incubated at 4°C overnight. The plates were washed three times with wash buffer (1X AquaLite® Wash Buffer (SeaLite Sciences, Inc., Bogart, GA) containing 0.3% Tween 20 (Sigma, St Louis, MO)), and blocked at 37°C for 1 h with
149
150 ml per well blocking buffer (1X Streptavidin AquaLite® Assay buffer (SeaLite Sciences, Inc., Bogart, GA) containing 5% goat serum). The plates were washed 3X and the test sera were diluted 1/300 or 1/9000 followed by serial threefold dilutions in the blocking buffer. A volume of 50 ml of each dilution was added per well and the plates were incubated at 37°C for 1 h. The plates were washed six times and incubated for 1 h at 37°C with 50 ml per well of Goat anti-mouse IgG-Biotin (Sigma St. Louis, MO), diluted 1/1000 in block buffer. After washing six times, the plates were incubated at 37°C for 1 h with Streptavidin– Aqualite® (SeaLite Sciences, Inc., Bogart, GA), diluted 1/500 in wash buffer, 50 ml per well. The plates were washed six times and stored in wash buffer until reactivity was measured on the luminometer (MLX, Dynex Technologies, Chantilly, VA). Setting for the luminometer—mode: Integrate Flash, Gain: High, Data: Table, Delay window: 0.00 s, Integrate window: 3.00 s, Before peak: 0.10 s, After peak: 2.00 s, calibrate on each well. The plates were tapped dry and put into the luminometer. Fifty microliters of 1X AquaLite® Trigger Buffer (SeaLite Sciences, Inc., Bogart, GA) were automatically dispensed per well and the relative light units (RLU) were measured. Endpoint titers were calculated as the inverse of the dilution that yields an RLU equal to the background plus five times the standard deviation.
3. Results and discussion Previous reports have demonstrated that application of electric current after injection of plasmid DNA has resulted in increased expression of the encoded proteins in the injected tissues (for example see Mathiesen, 1999; Mir et al., 1999). In this study, we used DNA plasmids encoding the reporter genes luciferase and b-galactosidase to measure transfection of muscles cells in vivo. At 4 days after a single inoculation of DNA, luciferase expression was higher in muscles treated with electric current compared to untreated muscles (Table 1). This was true for both iontophoresis (4.6-fold) and electroporation (7.3-fold). Simi-
150
M. Selby et al. / Journal of Biotechnology 83 (2000) 147–152
larly, the number of muscle fibers detectably transfected after inoculation of b-galactosidase DNA was substantially increased by iontophoresis and electroporation compared to untreated muscles, as judged by b-galactosidase staining of muscle tissue sections (data not shown). In addition, as noted previously (Mir et al., 1998), application of electric current appears to decrease the variability of reporter gene expression in muscle cells. Therefore, application of electric current facilitates delivery of DNA to muscle cells in situ and, hence, may be able to overcome some of the barriers to efficient transfection. To assess the effect of electric current on the immunogenicity of a DNA vaccine, mice received a single immunization with HIV gag DNA (10 mg) with or without electric current, and sera from mice were analyzed at 2, 4, 8 and 12 weeks after inoculation. At all time points tested, both iontophoresis and electroporation enhanced antibody titers in mice compared to those receiving no further treatment, with enhancement ranging from 8- to 20-fold (Fig. 1). As with luciferase expression levels, in general, electroporation conditions ap-
peared slightly superior to iontophoresis for enhancement of antibody responses. In a separate study, groups of mice were immunized twice with HIV gag DNA with a 6 week interval and antibody responses were measured after each immunization. Antibody titers were elevated in all groups after the booster injection, but the tenfold enhancement in titers observed in mice receiving electric current was maintained even after the boost (Fig. 2). It is not yet known how iontophoresis or electroporation increased DNA vaccine potency, but one reasonable possibility is that the distribution of DNA within the injected tissue and/or uptake of DNA by cells was facilitated by the electric current leading to increased transfection. The ensuing increase in mass of antigen expressed by cells likely played a role in the elevated immune responses. However, it is also possible that infiltration of inflammatory cells in response to the electric current could have taken place resulting in an ‘adjuvant’ effect on the antigen produced. Further work will be required to fully evaluate the mode of action of this delivery technology.
Table 1 Enhancement of gene expression in muscle cells in vivoa Treatment Luc DNA (no current) (10 mg)
Luc DNA (ionto) (10 mg)
Luc DNA (electro) (10 mg)
Luc activity 6.76 0.74 0.44 9.11 1.92 26.63 10.54 23.46 5.51 20.61 18.5 35.02 39.02 33.22 13.06
Mean (S.D.)
Fold increase
3.794 (3.91)
1.00
17.35 (8.95)
4.57
27.764 (11.30)
7.32
a Groups of five CB6 F1 mice were inoculated with 25 mg of luciferase (Luc) DNA in the TA muscle of one leg. One group of mice was not further treated, one group was treated with iontophoresis (ionto) and another with electroporation (electro). At 4 days after inoculation, the muscles were collected and luciferase activity was measured and expressed as ng luciferase activity per mg muscle protein. Numbers in brackets indicate standard deviation of the mean (S.D.).
M. Selby et al. / Journal of Biotechnology 83 (2000) 147–152
151
Fig. 1. Enhancement of antibody responses in mice. Groups of four to six CB6 F1 mice were inoculated a single time with 10 mg of HIV gag DNA in the TA muscle of one leg. One group of mice was not further treated, one group was treated with iontophoresis and another with electroporation. At the indicated intervals, sera were collected and analyzed for antibody responses. Data are plotted as geometric mean ELISA titer and error bars indicate S.E.M.
In summary, we have demonstrated that DNA vaccine potency can be increased by application of electric current, likely as a consequence of increased delivery of DNA to and into cells. These results indicate that a significant limitation to efficient transfection of cells in vivo by naked DNA vaccines is distribution within tissue and/or uptake of DNA by cells. It is not yet known if this limitation contributes to the lack of efficacy of DNA vaccines in larger animals, such as primates. If so, iontophoresis, electroporation and other means designed to facilitate delivery of DNA hold promise as second generation DNA vaccine technologies.
References
Fig. 2. Effect of boosting on antibody responses in mice. Groups of six CB6 F1 mice were inoculated with 10 mg of HIV gag DNA in the TA muscle of one leg at 3 and 6 weeks. One group of mice was not further treated (open bars), one group was treated with iontophoresis (solid bars) and another with electroporation (shaded bars). Sera were collected at 3 weeks after each immunization and analyzed for antibody responses. Data are plotted as geometric mean ELISA titer and error bars indicate S.E.M.
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