Skin targeted DNA vaccine delivery using electroporation in rabbits: I: Efficacy

doi:10.1016/j.ijpharm.2004.12.014

International Journal of Pharmaceutics

Volume 294, Issues 1–2, 27 April 2005, Pages 53-63

https://doi.org/10.1016/j.ijpharm.2004.12.014 Get rights and content

Abstract

Genetic immunization through skin is highly desirable as skin has plenty of antigen presenting cells (APCs) and is easily accessible. The purpose of this study was to investigate the effects of electroporation pulse amplitude, pulse length and number of pulses on cutaneous plasmid DNA vaccine delivery and immune responses, following intradermal injection in vivo in rabbits. Expression of the delivered plasmid was studied using a reporter plasmid, coding for β-galactosidase. The efficiency of DNA vaccine delivery was investigated using a DNA vaccine against Hepatitis B, coding for Hepatitis B surface antigen (HBsAg). Serum samples and peripheral blood mononuclear cells (PBMC) were analyzed for humoral and cellular immunity, respectively, following immunization. The expression of transgene in the skin was transient and reached its peak in 2 days post-delivery with 200 and 300 V pulses. The expression levels with 200 and 300 V pulses were 48- and 129-fold higher, respectively, compared with the passive on day 2. In situ histochemical staining of skin with X-gal demonstrated the localized expression of β-galactosidase with electroporation pulses of 200 and 300 V. Electroporation mediated cutaneous DNA vaccine delivery significantly enhanced both humoral and cellular immune responses (p < 0.05) to Hepatitis B compared to passive delivery. The present study demonstrates the enhanced DNA vaccine delivery to skin and immune responses by topical electroporation. Hence, electroporation mediated cutaneous DNA vaccine delivery could be developed as a potential alternative for DNA vaccine delivery.

Introduction

DNA vaccines offer a novel way to immunize individuals against deadly diseases using genetic material coding the antigens. The development of DNA vaccines is one of the most promising applications of recent advances in gene based therapy (Srivastava and Margaret, 2003). DNA vaccines are easy to produce, do not replicate and encode only the antigen of interest, as opposed to live attenuated or viral carrier systems. DNA vaccines express antigens in vivo, thus conserving the native conformation of the antigenic epitopes, which is critical for the induction of specific humoral antibody and cellular immune responses. The endogenously synthesized antigens can access both major histocompatibility complex (MHC) classes I and II pathways for antigen presentation (Leitner et al., 2000). DNA vaccines are shown to induce both cell mediated and humoral immunity, which are long lasting (Donnelly et al., 1997). They may be constructed to have more than one antigen-coding gene, thus potentially decreasing the number of vaccinations required.

Vaccination through skin is highly desirable as skin is most accessible and has a large pool of antigen presenting cells (APCs) that can process and present the antigen to appropriate lymphocytes efficiently. Skin contains plenty of functional bone marrow derived epidermal Langerhan's cells and dermal dendritic cells, which are specialized for induction of immune responses (Tuting et al., 1998). To be therapeutically useful, the DNA vaccine must be delivered inside the nucleus before it can express antigen molecules. This requires efficient membrane permeabilization to allow the DNA vaccines to enter the cells (Nishikawa and Huang, 2001). Following the report of gene expression after direct plasmid DNA injection (Wolff et al., 1990), several studies examined the possibility of vaccination using plasmid DNA coding antigens (DNA vaccines) in vivo (Ulmer et al., 1993, Raz et al., 1994, Lagging et al., 1995). Although direct injections of DNA vaccines do induce immune response in smaller animals, the delivery of the DNA to target cells is not optimal, especially in higher animals (Whalen, 1996, Waalen, 1997, Mumper and Ledebur, 2001). And also the amount of DNA used to induce immunity with direct injections is higher (Wang et al., 1998). The potency of immunization using DNA vaccines by topical route or intradermal injection has proven to be quite low (Donnelly et al., 1994, Fan et al., 1999, Shi et al., 1999). Several non-viral methods have been reported to enhance the immunogenicity of DNA vaccines, primarily by increasing the transfection efficiency. Most of these methods are not suitable for routine use either because of the safety concerns or due to the complex procedures and expenses involved (Herweijer and Wolff, 2003). Consequently, there remains a need to improve the skin targeted DNA vaccine delivery for efficient use of DNA vaccines for human immunization.

Recently, DNA vaccine delivery by electroporation has shown to be efficient (Selby et al., 2000, Widera et al., 2000, Zucchelli et al., 2000, Babiuk et al., 2002). Electroporation is a physical method, which involves application of short, high voltage electric pulses to permeabilize the target cell/tissue reversibly for macromolecules such as genes/proteins. Electroporation of cell membrane has been studied extensively and used for DNA transfection of the cells by reversibly permeabilizing the cell membranes (Neumann and Rosenheck, 1972, Kinosita and Tsong, 1977). The efficiency of transformation using electroporation is reported to be greater than the transformation efficiency by chemical methods (Andreason and Evans, 1988). Electroporation has been evaluated in animals and humans for the delivery of chemotherapeutic agents with high efficiency (Mir et al., 1998, Sersa et al., 2000). Furthermore, it has been employed in studies involving delivery of plasmid DNA in vivo to different types of tissues with improved transfection efficiency (Zhang et al., 1996, Muramatsu et al., 1997, Glasspool-Malone et al., 2000, Matsumoto et al., 2001, Blair-Parks et al., 2002, Liu and Huang, 2002). Electroporation mediated DNA vaccine delivery to muscle was shown to be a potential alternative to enhance the immunization efficiency (Widera et al., 2000, Zucchelli et al., 2000). But this procedure involves insertion of electrode needles into the muscle after intramuscular injection, which may not be feasible for use in humans. Cutaneous gene delivery using topical electroporation needs no specialized procedures, as the pulses would be applied topically with tweezer type of electrodes, following the injection of plasmid DNA. This was shown to be efficient for skin targeted gene transfer (Zhang et al., 2002).

Lack of safe and effective methods for delivering DNA vaccines may be the main reason for lower efficacy of these agents observed in higher animals and humans. This could be overcome by developing effective delivery methods that can improve the transfection and expression of DNA vaccines in vivo. In the present study, we investigated the expression and immune responses with skin targeted DNA vaccine delivery using topical electroporation for the delivery of DNA vaccine against Hepatitis B virus. The effects of electroporation pulse amplitude, pulse length and number were studied. Development of such a non-viral DNA vaccine delivery system is critical for the successful use of these agents for effective immunization against deadly infectious diseases.

Section snippets

Materials

The plasmid DNA coding for β-galactosidase, gWiz™ β-gal, and the plasmid coding for Hepatitis B surface antigen (HBsAg), pCMV-S (HBV DNA vaccine), were generously provided by Aldevron LLC (Fargo, ND, USA). The plasmids have an optimized human cytomegalovirus (CMV) promoter followed by intron A from the CMV immediate-early (IE) gene and a high efficiency polyadenylation transcription terminator. The purity of the plasmids was checked by measuring the ratio of A₂₆₀/A₂₈₀, which was higher than

Effect of electroporation pulse amplitude on cutaneous gene delivery

Table 1 shows the effect of electroporation pulse amplitude on the expression of β-galactosidase in the skin, 24 and 48 h after the cutaneous delivery of plasmid DNA. The reporter gene expression following the passive delivery (injection of plasmid without any pulses) and 100 V pulses was not significantly different (p > 0.05) from that of the control (no plasmid and no pulses). However, electroporation pulses of 200 and 300 V significantly enhanced (p < 0.05) the expression of β-galactosidase in

Discussion

Electroporation mediated gene delivery is shown to be highly efficient in various studies with different mammalian cells in vitro (Toneguzzo and Keating, 1986, Andreason and Evans, 1988, Espinos et al., 2001). Recently, this method has been shown to be effective for gene transfer in vivo in many types of tissues including the skin (Zhang et al., 1996, Muramatsu et al., 1997, Glasspool-Malone et al., 2000, Matsumoto et al., 2001, Blair-Parks et al., 2002, Liu and Huang, 2002). Most of these in

Conclusions

The major problem with the non-viral gene transfer is the low levels of transgene expression. The present study demonstrates that the injection of naked plasmid DNA followed by topical electroporation improves transfection efficiency in vivo by several folds, especially after a threshold voltage. The results show the efficacy of skin targeted DNA vaccine delivery using electroporation to induce strong humoral as well as cellular immunity against a disease. Although the expression of the

Acknowledgements

We acknowledge the financial support from National Institutes of Health grant #HD 41372 and the Presidential Doctoral Fellowship to B.M.M. from North Dakota State University, Fargo, ND 58105. The generous supply of plasmid DNA by Aldevron L.L.C. is thankfully acknowledged. We also thank Somnath Singh and Sumeet Rastogi for their help during in vivo experimentation.

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Skin targeted DNA vaccine delivery using electroporation in rabbits: I: Efficacy

Abstract

Introduction

Section snippets

Materials

Effect of electroporation pulse amplitude on cutaneous gene delivery

Discussion

Conclusions

Acknowledgements

Vaccine

J. Immunol. Methods

Neuromuscul. Disord.

Mol. Ther.

Biochem. Biophys. Res. Commun.

Bioelectrochem. Bioenerg.

Biophys. J.

J. Biotechnol.

Vaccine

J. Invest. Dermatol.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Gene transfer into muscle by electroporation in vivo

Nat. Biotechnol.

Liposome-mediated gene transfer and expression via the skin

Hum. Mol. Genet.

Introduction and expression of DNA molecules in eukaryotic cells by electroporation

Biotechniques

High-level gene transfer to the cornea using electroporation

J. Gene Med.

DNA vaccines

Annu. Rev. Immunol.

Topical gene transfer into rat skin using electroporation

Pharm. Res.

Immunization via hair follicles by topical application of naked DNA to normal skin

Nat. Biotech.

Progress and prospects: naked DNA gene transfer and therapy

Gene Ther.