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Genetic immunization by jet injection of targeted pDNA-coated nanoparticles
Methods 31 (2003) 255–262 www.elsevier.com/locate/ymeth
Genetic immunization by jet injection of targeted pDNA-coated nanoparticles Russell J. Mumper* and Zhengrong Cui Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 907 Rose Street, Lexington, KY 40536-0082, USA Accepted 25 April 2003
Abstract Genetic immunization strategies have largely focused on the use of ‘‘naked’’ plasmid DNA or the gene gun. However, there remains a clear need to further improve the efficiency and/or cost of potential DNA vaccines. The theoretical basis of our research is to rationally design genetic immunization methodologies for nanoparticle-based delivery systems of plasmid DNA, perhaps in combination with already commercially available needle-free devices, such as the Biojector 2000. These methodologies may both reduce the dose of pDNA required and enhance the breadth and depth of protective immune responses (i.e., humoral and cellular). The purpose of this article is to provide detailed experimental methods to (1) engineer and characterize pDNA-coated cationic nanoparticles (<100 nm) directly from oil-in-water microemulsion precursors and (2) enhance both the breadth and depth of immune responses after immunization of mice with pDNA-coated nanoparticles by different routes of administration, including intradermal, using a needle-free jet injection device. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: DNA vaccine; Microemulsion; b-Galactosidase; Mannan
1. Introduction Genetic immunization with plasmid DNA (pDNA) to express antigens has emerged as a clinically viable and potentially safe vaccination strategy. To date, the most common methods of genetic immunization involve either intramuscular injection of ‘‘naked’’ plasmid DNA or delivery of pDNA via gene gun (e.g., ballistic administration of plasmid DNA-laden gold beads) [1]. The Helios (previously called Accell) gene gun system (BioRad, Hercules, CA) is available only for experimental purposes. A derivative device permitted for clinical use is available from PowderJect. Both apparatuses require the preparation of ‘‘backshot,’’ DNA-coated gold beads, which are fired by compressed gas (see also Sasaki et al., this issue). However, both gene gun and needle injections have limitations. Immunization with naked plasmid DNA requires very large doses. For example, a report by Wang et al. [2] described the results * Corresponding author. Fax: 1-859-323-5985. E-mail address: [email protected] (R.J. Mumper).
of a human clinical trial of a malaria DNA vaccine where three 2.5 mg doses of pDNA were administered. Additional human clinical trials using pDNA administered by intramuscular needle injection showed that cytotoxic T-lymphocyte (CTL) responses were elicited [3] but not antibodies to expressed antigen [4]. In contrast, clinical trials using the PowderJect gene gun device (Madison, WI) to administer DNA vaccines to the skin epidermis in humans showed that all the subjects (total of 11) seroconverted to antibody levels, which are known to confer protection against hepatitis B [5]. However, it is unclear whether or not this device and technology may be commercially viable. Thus, we need to improve the efficiency of DNA vaccines. The theoretical basis of our research is to rationally design genetic immunization methodologies founded on nanoparticle-based delivery systems for plasmid DNA, perhaps in combination with already commercially available needle-free devices, such as the Biojector 2000. These methodologies may both reduce the dose of pDNA required and enhance the breadth and depth of protective immune responses (i.e., humoral and cellular).
1046-2023/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-2023(03)00138-5
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Needle-free jet injection has been investigated extensively as a method to immunize laboratory animals, such as mice [6,7], rabbits [8–10], pigs and dogs [11], and monkeys [7,12]. In addition, jet injection has been tested in several human clinical trials [2,13–22]. The vast majority of the studies in animals have demonstrated an enhancement in the resulting immune responses with jet injection over needle and syringe. For example, Aguiar et al. [10] immunized rabbits with three 500 lg doses of a plasmid encoding malarial antigen (Plasmodium falciparum circumsporozoite protein, pfCSP) by intramuscular needle injection, intramuscular jet injection (Biojector 2000), and a combination of intramuscular and intradermal jet injections. An indirect fluorescence antibody test demonstrated pfCSP titers that were 8– 50-fold greater with jet injection than with intramuscular needle injection. Moreover, both routes of immunization by jet injection resulted in seroconversion, whereas rabbits immunized by intramuscular needle injection failed to seroconvert. In another study, Anwer et al. [11] immunized both pigs and dogs with a plasmid expressing human growth hormone (hGH) and compared subcutaneous and intramuscular needle injections to jet injection (Medi-Jector). The results showed an enhancement in antigen-specific titer that ranged 3–20-fold over needle injection by either route, depending on the pDNA formulation used. Finally, our laboratory demonstrated significant enhancements in antibody responses (IgG and IgA) in mice after jet injection (Biojector 2000) of pDNA-coated nanoparticles over needle injection [6]. For example, jet injection of pDNA-coated nanoparticles led to as high as a 20-fold enhancement in IgG titer over subcutaneous needle injection of the same nanoparticles. In addition, jet injection of pDNA-coated nanoparticles enhanced the IgG titer by more than 200-fold over jet injection of pDNA alone. Although the majority of the studies in animals reporting that jet injection potentiates immune responses, this is not always the case in human trials. For example, two clinical studies have shown that jet injection enhanced the rate of seroconversion but not the extent [14,21]. Three additional clinical studies demonstrated no statistical benefit in enhancing immune responses by jet injection [15,20,22]. However, at least six human studies demonstrated comparable or enhanced immune responses using jet injection over needle injection [2,13,16–19]. It remains difficult to draw a conclusion about the benefits of jet injection over needle injection from the literature because the reports described above involved many different variables, such as type of vaccine, jet injection device, route, dose, antigen(s), dose schedule, and disease model. It is generally agreed that jet injection may be used more easily and safely to consistently administer vaccines to a large population, which may make widespread immunization initiatives more feasi-
ble. The mechanism of enhancement of immune response with jet injection remains largely unknown; however, it has been reported that jet injection is able to produce a larger distribution pattern of the injectate at the injection site compared with the traditional needleand-syringe injection [23–25]. This enhanced distribution may more efficiently target immune cells [7,24–26]. Another aspect of jet injection that may aid in the enhancement of immune response is the higher incidence of local but transient inflammation that has been reported after jet injection, compared with injection of needle and syringe [13,15–17,21]. This transient effect may help to recruit inflammatory/immune cells to the injection site, which may enhance the resulting immune response. Formulation and delivery approaches may also be used to improve the efficacy of genetic immunization. For example, the potential of nanoparticles and microspheres to enhance immune responses after pDNA delivery has been explored extensively [27]. We described a method to engineer cationic nanoparticles (<100 nm) directly by cooling of warm oil-in-water microemulsion precursors [6,28–32]. We coated plasmid DNA on the surface of cationic nanoparticles (pDNA-coated nanoparticles). An endosomolytic lipid (dioleoyl phosphatidylethanolamine, DOPE) and a dendritic cell (DC)-specific ligand (mannan) were successfully incorporated in, or attached on the surface of, the nanoparticles. Immunization of mice with pDNA-coated nanoparticles by the subcutaneous [28], topical [29], and intranasal routes [30] or intradermal administration with jet injection [6] led to enhanced immune responses of a model antigen, b-galactosidase, as compared with immunization of naked pDNA alone. These nanoparticles were combined with other known adjuvants, such as cholera toxin and Lipid A, to further enhance the resulting immune responses [32]. Here, we provide detailed experimental methods of our work with engineered dendritic cell-targeted pDNAcoated nanoparticles for enhanced genetic immunization.
2. Description of methods 2.1. Engineering of cationic nanoparticles 1. Melt emulsifying wax (2 mg) at 55 °C in 7 mL glass vials. Add warm water (0.7 mL; 0.2 lm filtered) to the melted wax and stir with a 1=4 in. magnetic stir bar at 55 °C until a homogeneous milky suspension results. 2. Add 0.3 mL CTAB solution (50 mM) into the homogenate while stirring to obtain a clear microemulsion. Cool the warm microemulsion to room temperature in the same glass vial during stirring to engineer nanoparticles.
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3. For the incorporation of an endosomolytic agent, mix 100 lg DOPE (final 5% w/w) with the emulsifying wax (2 mg), prior to microemulsion preparation. Previous studies showed that the incorporation of DOPE into the nanoparticles significantly enhanced the in vitro transfection efficiency of pDNA-coated nanoparticles, as well as the resulting immune responses after different routes of administration [6,28–32]. 2.2. Deposition of cholesterol-mannan (chol-mannan) on the surface of the nanoparticles Human and mouse dendritic cells have been shown to express a mannose receptor [33,34]. Mannan, a 42 kDa Mw polysaccharide comprising 233 mannose residues, has been previously used to target DCs and enhance immune responses [35,36]. Cholesterol-modified mannan (chol-mannan) was used to anchor the mannan at the surface of the cured cationic nanoparticles. 1. Mix 1 mL of the pre-formed nanoparticle suspension (2 mg/mL) with 250 lg of chol-mannan, previously dissolved in hot water (5 mg/mL), and stir at room temperature overnight. 2. Alternatively, add chol-mannan directly to the warm oil-in-water microemulsion for similar results. This method eliminates overnight stirring. 2.3. Purification of nanoparticles by gel permeation chromatography 1. Soak Sephadex G-75 (powder) in de-ionized water for 24 h at room temperature prior to column preparation. 2. Remove free CTAB and chol-mannan by passing the nanoparticle suspension through a Sephadex G-75 column (14 65 mm) with water or 10% lactose as the mobile phase. Free CTAB can be easily removed from cationic nanoparticles by gel permeation chromatography (GPC) with no significant effect on cationic nanoparticle size. The loading volume of the nanoparticle suspension should be minimized to obtain better separation. 3. After GPC purification, pass nanoparticles through a 0.22 lm filter for sterilization.
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the surface of the nanoparticles before performing particle size and zeta potential measurements. 2.5. Particle size and zeta potential measurements of nanoparticles 2.5.1. Particle size 1. Prior to particle sizing, dilute the nanoparticle suspensions with water (0.2 lm filtered), so that the signal from the instrument, measured in counts per second (CPS), lies within the sensitivity range of 50,000 to 1 106 CPS. 2. Measure the particle sizes with an N4 Plus Sub-Micron Particle Sizer (Coulter, Miami, FL). Use plastic cuvettes (Beckman Coulter, Fullerton, CA; Cat. No. 7800091) at 25 °C for 120 s, scattering light at 90 °. As shown in Fig. 1, the particle size of mannancoated cationic nanoparticles was approximately 100 nm, whereas the coating of plasmid DNA resulted in about a 2.7-fold increase in particle size to about 270 nm. 2.5.2. Zeta potential Measure the zeta potential of the nanoparticles using a Zeta Sizer 2000 (Malvern Instruments, Southborough, MA). Mannan-coated cationic nanoparticles had a positive zeta potential of +41 mV. Upon coating with pDNA, the zeta potential decreased to +32 mV, as expected (Fig. 1). 2.6. Transmission electron microscopy We observed the size and morphology of nanoparticles cured from microemulsion precursors using a
2.4. Coating of plasmid DNA on the surface of the nanoparticles 1. Coat plasmid DNA (cytomegalovirus [CMV]-b-galactosidase) on the surface of the nanoparticles by gently mixing 1 mL of the purified and filter-sterilized nanoparticles in suspension with pDNA to obtain a final pDNA concentration of 50 lg/mL. 2. Let the formulation remain at least 30 min at room temperature for complete adsorption of pDNA to
Fig. 1. Particle size and zeta potential of mannan-coated cationic nanoparticles (Man-NPs) and pDNA-coated nanoparticles (Man-NPs with pDNA). The addition of pDNA to the mannan-coated nanoparticles caused an increase in the particle size (white bars) and a decrease in the zeta potential (filled circles), as expected.
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Fig. 2. Transmission electron micrographs. (A) Mannan-coated cationic nanoparticles and (B) pDNA-coated nanoparticles. Spherical and homogeneous cationic nanoparticles were engineered directly from the oil droplets of a clear microemulsion composed of emulsifying wax (2 mg), DOPE (100 lg), and CTAB (15 mM) in 1 mL water at 55 °C, cooling to room temperature during gentle stirring. Chol-mannan was added as described in Section 2. Mannan-coated cationic nanoparticles were purified by gel permeation chromatography, prior to the addition of pDNA. Plasmid DNA (CMV-b-galactosidase) was coated on the surface of the cationic nanoparticles (1 mL) to obtain a final pDNA concentration of 50 lg/mL.
Philips Tecnai 12 Transmission Electron Microscope in the Imaging Facility Unit, University of Kentucky. 1. On a carbon-coated 200-mesh copper specimen grid, glow-discharge the grid for 1.5 min. Deposit one drop of nanoparticle suspension on the grid and allow to stand for 1.5 min. Remove any excess fluid with filter paper. 2. Stain the grid with 1 drop of 1% uranyl acetate (0.2 lm filtered) and allow to dry for at least 30 min before examining under the electron microscope. As shown in Fig. 2A, the cationic nanoparticles were spherical and uniform, having an average particle size of 100 nm, which agreed with the results obtained by laser light scattering. Examination of the pDNA-coated nanoparticles showed surface pDNA as depicted by the darker staining pattern (Fig. 2B). 2.7. In vitro ConA agglutination assay Concanavalin A (ConA) is a plant lectin, a tetrameric protein with four binding sites specific for terminal mannosyl residues. Binding to the terminal a-D mannosyl residues of mannan will cause agglutination (or aggregation) of the complex in solution, resulting in an increase in turbidity. 1. Add increasing amounts of chol-mannan (standard) or mannan-coated nanoparticles (200 lL) at room temperature to 1 mL ConA (1 mg/mL) in 10 mM phosphate-buffered saline, pH 7.4, with 5 mM calcium chloride and 5 mM magnesium chloride. 2. Monitor the increase in turbidity at 360 nm for 200 s using a U-2000 Spectrophotometer (Hitachi Instruments; Danbury, CT). As shown in Fig. 3, mannan-coated nanoparticles with and without pDNA caused significant agglutination of ConA over 200 s, whereas both uncoated nanoparticles and free pDNA did not.
Fig. 3. Concanavalin A (ConA) agglutination assay to confirm the presence of mannan on the surface of mannan-coated cationic nanoparticles (Man-NPs) or pDNA-coated nanoparticles (Man-NPs with pDNA). Samples (0.2 mL) were added to 1 mL ConA (1 mg/mL) and the increase in turbidity at 360 nm was measured for 200 sec and compared with a ConA standard curve (insert). Control samples, either cationic nanoparticles (free NPs) or pDNA (free pDNA), did not cause ConA agglutination.
2.8. Immunization of mice We used 10- to 12-week-old female mice (Balb/C; Harlan Sprague–Dawley Laboratories, Indianapolis, IN) for all animal studies, observing NIH guidelines for the care and use of laboratory animals. 1. Anesthetize a mouse using pentobarbital (60 mg/kg body weight) by intraperitoneal injection. 2. On days 0, 7, and 14, immunize the mouse (n ¼ 5–6/ group) either with naked pDNA alone (5 lg), pDNA (5 lg)-coated nanoparticles, or b-galactosidase protein (10 lg) with the adjuvant Alum (Spectrum, 15 lg). 2.8.1. Topical immunization (derived from [29,32,37,38]) 1. Shave the hair on the back of the mouse with an A5 Single-Speed Clipper (Oster Professional Products,
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McMinnville, TN). Wipe the skin with an alcohol swab and allow to air dry. (One does not need to shave the hair for subcutaneous injections.) 2. Spread 100 lL of each formulation with a pipette tip onto the skin, covering an area of about 2 cm2 . Carefully disperse the solution over the skin without applying pressure.
2. Place the mouse on a 1-in.-thick foam pad, making sure the hind legs are moved from the abdomen underneath the mouse. Anesthetize by intraperitoneal pentobarbital injection as described above. 3. Position the Biojector 2000 device with a #2 intradermal spacer perpendicularly to the back of the mouse. Release the injectate (100 lL) carefully into the skin.
2.8.2. Subcutaneous immunization (derived from [28,31,32]) Briefly, pull the skin on the back of the neck upward to create a pinch of skin. Carefully insert the needle into the pinched skin and inject 100 lL of each formulation (in 10% lactose) with an insulin syringe with MICROFINE IV Needle (Becton–Dickinson, Franklin Lakes, NJ) on one site on the back, creating a bubble of liquid in the subcutaneous space.
2.9. Collection of biological samples
2.8.3. Jet injection immunization (derived from [6]) We used the Biojector 2000 needle-free jet injection device (Bioject, Portland, OR; Fig. 4), following the procedure for intradermal delivery in rats and mice. The Biojector 2000 delivers a titrated volume of liquid as a fine spray into the epidermis. The spray is driven by compressed carbon dioxide (CO2 ) gas contained in a small cylinder in the hand-held device (see www.bioject.com). 1. Shave a mouse (1–2 cm2 ) on the back area about halfway up starting at the base of the tail. Swab the shaved area thoroughly with 70% alcohol swab and allow to air dry.
2.9.1. Terminal blood collection 1. On day 28, anesthetize the mouse by intraperitoneal pentobarbital injection as described above. 2. For bleeding by cardiac puncture, tape the forelimbs of the mouse separately at a 90° angle to the body. Place a piece of tape across the lower abdomen of the mouse. 3. Insert a 21G 1 in. needle (with a 1 mL syringe) at an approximately 30° angle, immediately caudal to the xyphoid process. Aim the needle toward the left shoulder. Collect approximately 0.4–0.5 mL of blood. 4. Transfer the blood to a Vacutainer Brand Blood Collection Tube (Becton–Dickinson). Separate the serum centrifugation and store at )20 °C for analysis. 2.9.2. Spleen removal and preparation of splenocytes 1. Remove spleens from mice on day 28 and place in 5 mL HanksÕ Balanced Salt Solution (HBSS) (1) in a Stomacher Bag 400 (Fisher Scientific, Pittsburgh, PA). Homogenize the spleens at high speed for 60 s using a Stomacher Homogenizer.
Fig. 4. The Biojector 2000 (Bioject, Portland, OR) is a small handheld device that is used for needle-free delivery of antigen. It delivers a volume of liquid as a fine spray into the epidermis, the spray is driven by compressed carbon dioxide (CO2 ) gas contained in a small cylinder within the actual device itself.
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2. Transfer cell suspensions into 15 mL Falcon tubes and fill to 15 mL with 1 ACK buffer (156 mM NH4 Cl, 10 mM KHCO3 , and 100 lM EDTA) for red blood cell lysis. 3. After 5–8 min at room temperature, spin down the suspension 1500 rpm for 7 min at 4 °C. After pouring off the supernatant, re-suspend the cell pellet in 15 mL HBSS. Spin down the suspension at 1500 rpm for another 7 min at 4 °C. 4. After washing with 15 mL RPMI-1640 (BioWhittaker, Walkersville, MD), supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO) and 0.05 mg/mL gentamicin (Gibco-BRL, Gaithersburg, MD), re-suspend the cells in RPMI 1640 media (1 mL for each spleen in the pool). 2.10. Determination of antibody titers 1. To quantify b-galactosidase-specific serum antibody titers by ELISA, begin by coating Costar high binding 96-well assay plates with 50 lL b-galactosidase protein (8 lg/mL) overnight at 4 °C. Block the plates for 1 h at 37 °C with 4% bovine serum albumin (BSA)/ 4% NGS (Sigma) solution (100 lL/well) made in 1 PBS/Tween 20 (ScyTek Laboratories, Logan, UT). 2. Serially dilute mouse serum (50 lL/well) in 4% BSA/ 4% NGS/PBS/Tween 20 and incubate for 2 h at 37 °C. After washing three times with PBS/Tween 20 buffer, add anti-mouse IgG HRP F(ab0 )2 fragment from sheep (diluted 1:3000 in 1% BSA, 50 lL/well) and incubate for 1 h at 37 °C. Wash plates three times with PBS/Tween 20 buffer. Finally, develop the samples with 100 lL TMB substrate for 30 min at room temperature and stop with 50 lL of 0.2 M H2 SO4 . 3. Measure the OD of each well using a Universal Microplate Reader (Bio-Tek Instruments, Winooski, VT) at 450 nm. Assign the titer the highest dilution at which the OD450 is 2 SDs higher than that of the na€ıve group at equivalent dilutions. 4. Determine the antigen-specific serum IgA titer using a method similar to the IgG ELISA with modification. Specifically, serial dilute the serum samples at 1:5 in 4% BSA/4% NGS/PBS/Tween 20. In addition, peroxidase-linked goat anti-mouse IgA (1:6000 in 1% BSA) was used. Intradermal jet injection of pDNA-coated nanoparticles led to the highest antibody titer of all groups by any route with a geometric mean titer of over 160,000 (Fig. 5). The titer obtained with pDNA-coated nanoparticles delivered by jet injection was over 20-fold and 65-fold greater than that in mice immunized with pDNA-coated nanoparticles given by the subcutaneous and topical routes, respectively (p < 0:05). For naked pDNA alone, there was no significant difference in specific serum IgG titer (p ¼ 0:46) after jet injection and subcutaneous immunization, and both titers were sig-
Fig. 5. b-Galactosidase-specific IgG titer in the sera of mice at day 28. Mice were dosed on days 0, 7, and 14 with 5 lg pDNA in the form of naked pDNA (pDNA) or pDNA-coated nanoparticles (Man-NPs), or with b-galactosidase protein (10 lg) containing alum (15 lg) (bgal + alum). Data shown are the geometric mean titer with standard deviation (n ¼ 5–6) [6,28,29].
nificantly greater than after topical application of naked pDNA. By the subcutaneous route, the specific IgG titer in the sera of the mice immunized with pDNA-coated nanoparticles was 10-fold higher than that of the mice immunized with naked pDNA alone (p ¼ 0:039). After intradermal jet injection, the specific serum IgG titer was enhanced by more than 200-fold when mice were immunized with pDNA-coated nanoparticles, compared with jet injection with naked pDNA alone (p < 0:05). Immunization with pDNA-coated nanoparticles by jet injection led to more than a log increase in the IgA titer (mean of 800) elicited with naked pDNA given by either jet injection (p ¼ 0:047) or subcutaneous injection (p ¼ 0:072). Immunization with b-galactosidase protein formulated with Alum and administered by both jet and subcutaneous injection led to antigen-specific IgG and IgA titers, although the titers were statistically lower than those obtained by immunization with pDNAcoated nanoparticles. The dose of b-galactosidase protein and/or dosing regimen may have contributed, in part, to these low titers. 2.11. In vitro cytokine release and splenocyte proliferation We performed in vitro cytokine release assays and splenocyte proliferation assays using isolated splenocytes from immunized mice as described in previous reports [6,28–32]. By all routes of administration, pDNA-coated nanoparticles significantly enhanced Th1-type cytokine (IFN-c) production and splenocyte proliferative responses over naked pDNA alone. For example, immunization with pDNA-coated nanoparticles led to a
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5.6-fold enhancement in IFN-c production by jet injection (p ¼ 0:026) and a 2.8-fold enhancement by subcutaneous injection (p ¼ 0:006), compared with naked pDNA alone. Similarly, the pDNA-coated nanoparticles led to a 3.5-fold enhancement in IL-4 release by jet injection (p ¼ 0:03) and 2-fold enhancement by subcutaneous injection (p ¼ 0:07), compared with naked pDNA alone. Immunization with pDNA-coated nanoparticles resulted in a more balanced Th1/Th2 immune response by all routes of administration. Administration of naked pDNA alone did not have a significant effect on cytokine release from isolated splenocytes or in the proliferation of the splenocytes. 2.12. Statistical analyses We completed all statistical analyses using a one-way ANOVA, followed by pair-wise comparisons with FisherÕs protected least significant difference procedure, unless mentioned otherwise. A p value of 6 0.05 was considered to be statistically significant.
3. Conclusions Our research aims to rationally design genetic immunization methodologies based on nanoparticle delivery systems for plasmid DNA in combination with already available needle-free devices, such as the Biojector 2000. These methodologies may both reduce the dose of pDNA required and enhance the breadth and depth of protective immune responses (i.e., humoral and cellular). Methods have been developed to combine dendritic cell-targeted pDNA-coated nanoparticles with a needle-free device to significantly enhance antibody and Th1-type immune responses. The results from these studies demonstrated that a combination of improved pDNA delivery systems and an alternative route of administration significantly enhanced the breadth and depth of immune responses from a DNA vaccine.
Acknowledgments The authors thank Dr. Lawrence Baizer at Bioject Medical Technologies (Portland, OR) for his helpful comments and instruction on the use of the Biojector 2000.
Appendix. List of key materials Plasmid containing a CMV promoter with b-galactosidase reporter gene (CMV-b-gal) with endotoxin <0.1 EU/mg (Valentis, Woodlands, TX)
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Emulsifying wax (N.F. grade) (Spectrum Quality Products, New Brunswick, NJ) Aluminum hydroxide gel (Alum) (Spectrum Quality Products, New Brunswick, NJ) Hexadecyltrimethyl-ammonium bromide (CTAB) (Sigma Chemical, St. Louis, MO) b-Galactosidase protein (Sigma Chemical) Normal goat serum (NGS) (Sigma Chemical) Bovine serum albumin (BSA) (Sigma Chemical) Sephadex G-75 (Sigma Chemical) Phosphate-Buffered Saline/Tween 20 buffer (20) (ScyTek Laboratories, Logan, UT) Anti-mouse IgG, peroxidase-linked species-specific F(ab0 )2 fragment (from sheep) (Amersham–Pharmacia Biotech, Piscataway, NJ) Goat anti-mouse IgA, peroxidase-linked (Southern Biotechnology Associates, Birmingham, AL) Tetramethylbenzidine (TMB) (Pierce, Rockford, IL) Dioleoylphosphatidylethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL) {N-[2-(Chloesterylcarboxyamino)ethyl]carbamoylmethyl}mannan (chol-mannan) (Dojindo Molecular Technologies, Gaithersburg, MD) Mouse Interleukin-4 (IL-4) ELISA kit (Pierce-Endogen, Rockford, IL) Interferon-c (IFN-c) ELISA kit (Pierce-Endogen) CellTiter 96 Aqueous non-radioactive cell proliferation assay kit (Promega, Madison, WI) Concanavalin A (ConA) (Sigma Chemical)
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Genetic immunization by jet injection of targeted pDNA-coated nanoparticles Russell J. Mumper* and Zhengrong Cui Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 907 Rose Street, Lexington, KY 40536-0082, USA Accepted 25 April 2003
Abstract Genetic immunization strategies have largely focused on the use of ‘‘naked’’ plasmid DNA or the gene gun. However, there remains a clear need to further improve the efficiency and/or cost of potential DNA vaccines. The theoretical basis of our research is to rationally design genetic immunization methodologies for nanoparticle-based delivery systems of plasmid DNA, perhaps in combination with already commercially available needle-free devices, such as the Biojector 2000. These methodologies may both reduce the dose of pDNA required and enhance the breadth and depth of protective immune responses (i.e., humoral and cellular). The purpose of this article is to provide detailed experimental methods to (1) engineer and characterize pDNA-coated cationic nanoparticles (<100 nm) directly from oil-in-water microemulsion precursors and (2) enhance both the breadth and depth of immune responses after immunization of mice with pDNA-coated nanoparticles by different routes of administration, including intradermal, using a needle-free jet injection device. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: DNA vaccine; Microemulsion; b-Galactosidase; Mannan
1. Introduction Genetic immunization with plasmid DNA (pDNA) to express antigens has emerged as a clinically viable and potentially safe vaccination strategy. To date, the most common methods of genetic immunization involve either intramuscular injection of ‘‘naked’’ plasmid DNA or delivery of pDNA via gene gun (e.g., ballistic administration of plasmid DNA-laden gold beads) [1]. The Helios (previously called Accell) gene gun system (BioRad, Hercules, CA) is available only for experimental purposes. A derivative device permitted for clinical use is available from PowderJect. Both apparatuses require the preparation of ‘‘backshot,’’ DNA-coated gold beads, which are fired by compressed gas (see also Sasaki et al., this issue). However, both gene gun and needle injections have limitations. Immunization with naked plasmid DNA requires very large doses. For example, a report by Wang et al. [2] described the results * Corresponding author. Fax: 1-859-323-5985. E-mail address: [email protected] (R.J. Mumper).
of a human clinical trial of a malaria DNA vaccine where three 2.5 mg doses of pDNA were administered. Additional human clinical trials using pDNA administered by intramuscular needle injection showed that cytotoxic T-lymphocyte (CTL) responses were elicited [3] but not antibodies to expressed antigen [4]. In contrast, clinical trials using the PowderJect gene gun device (Madison, WI) to administer DNA vaccines to the skin epidermis in humans showed that all the subjects (total of 11) seroconverted to antibody levels, which are known to confer protection against hepatitis B [5]. However, it is unclear whether or not this device and technology may be commercially viable. Thus, we need to improve the efficiency of DNA vaccines. The theoretical basis of our research is to rationally design genetic immunization methodologies founded on nanoparticle-based delivery systems for plasmid DNA, perhaps in combination with already commercially available needle-free devices, such as the Biojector 2000. These methodologies may both reduce the dose of pDNA required and enhance the breadth and depth of protective immune responses (i.e., humoral and cellular).
1046-2023/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-2023(03)00138-5
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Needle-free jet injection has been investigated extensively as a method to immunize laboratory animals, such as mice [6,7], rabbits [8–10], pigs and dogs [11], and monkeys [7,12]. In addition, jet injection has been tested in several human clinical trials [2,13–22]. The vast majority of the studies in animals have demonstrated an enhancement in the resulting immune responses with jet injection over needle and syringe. For example, Aguiar et al. [10] immunized rabbits with three 500 lg doses of a plasmid encoding malarial antigen (Plasmodium falciparum circumsporozoite protein, pfCSP) by intramuscular needle injection, intramuscular jet injection (Biojector 2000), and a combination of intramuscular and intradermal jet injections. An indirect fluorescence antibody test demonstrated pfCSP titers that were 8– 50-fold greater with jet injection than with intramuscular needle injection. Moreover, both routes of immunization by jet injection resulted in seroconversion, whereas rabbits immunized by intramuscular needle injection failed to seroconvert. In another study, Anwer et al. [11] immunized both pigs and dogs with a plasmid expressing human growth hormone (hGH) and compared subcutaneous and intramuscular needle injections to jet injection (Medi-Jector). The results showed an enhancement in antigen-specific titer that ranged 3–20-fold over needle injection by either route, depending on the pDNA formulation used. Finally, our laboratory demonstrated significant enhancements in antibody responses (IgG and IgA) in mice after jet injection (Biojector 2000) of pDNA-coated nanoparticles over needle injection [6]. For example, jet injection of pDNA-coated nanoparticles led to as high as a 20-fold enhancement in IgG titer over subcutaneous needle injection of the same nanoparticles. In addition, jet injection of pDNA-coated nanoparticles enhanced the IgG titer by more than 200-fold over jet injection of pDNA alone. Although the majority of the studies in animals reporting that jet injection potentiates immune responses, this is not always the case in human trials. For example, two clinical studies have shown that jet injection enhanced the rate of seroconversion but not the extent [14,21]. Three additional clinical studies demonstrated no statistical benefit in enhancing immune responses by jet injection [15,20,22]. However, at least six human studies demonstrated comparable or enhanced immune responses using jet injection over needle injection [2,13,16–19]. It remains difficult to draw a conclusion about the benefits of jet injection over needle injection from the literature because the reports described above involved many different variables, such as type of vaccine, jet injection device, route, dose, antigen(s), dose schedule, and disease model. It is generally agreed that jet injection may be used more easily and safely to consistently administer vaccines to a large population, which may make widespread immunization initiatives more feasi-
ble. The mechanism of enhancement of immune response with jet injection remains largely unknown; however, it has been reported that jet injection is able to produce a larger distribution pattern of the injectate at the injection site compared with the traditional needleand-syringe injection [23–25]. This enhanced distribution may more efficiently target immune cells [7,24–26]. Another aspect of jet injection that may aid in the enhancement of immune response is the higher incidence of local but transient inflammation that has been reported after jet injection, compared with injection of needle and syringe [13,15–17,21]. This transient effect may help to recruit inflammatory/immune cells to the injection site, which may enhance the resulting immune response. Formulation and delivery approaches may also be used to improve the efficacy of genetic immunization. For example, the potential of nanoparticles and microspheres to enhance immune responses after pDNA delivery has been explored extensively [27]. We described a method to engineer cationic nanoparticles (<100 nm) directly by cooling of warm oil-in-water microemulsion precursors [6,28–32]. We coated plasmid DNA on the surface of cationic nanoparticles (pDNA-coated nanoparticles). An endosomolytic lipid (dioleoyl phosphatidylethanolamine, DOPE) and a dendritic cell (DC)-specific ligand (mannan) were successfully incorporated in, or attached on the surface of, the nanoparticles. Immunization of mice with pDNA-coated nanoparticles by the subcutaneous [28], topical [29], and intranasal routes [30] or intradermal administration with jet injection [6] led to enhanced immune responses of a model antigen, b-galactosidase, as compared with immunization of naked pDNA alone. These nanoparticles were combined with other known adjuvants, such as cholera toxin and Lipid A, to further enhance the resulting immune responses [32]. Here, we provide detailed experimental methods of our work with engineered dendritic cell-targeted pDNAcoated nanoparticles for enhanced genetic immunization.
2. Description of methods 2.1. Engineering of cationic nanoparticles 1. Melt emulsifying wax (2 mg) at 55 °C in 7 mL glass vials. Add warm water (0.7 mL; 0.2 lm filtered) to the melted wax and stir with a 1=4 in. magnetic stir bar at 55 °C until a homogeneous milky suspension results. 2. Add 0.3 mL CTAB solution (50 mM) into the homogenate while stirring to obtain a clear microemulsion. Cool the warm microemulsion to room temperature in the same glass vial during stirring to engineer nanoparticles.
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3. For the incorporation of an endosomolytic agent, mix 100 lg DOPE (final 5% w/w) with the emulsifying wax (2 mg), prior to microemulsion preparation. Previous studies showed that the incorporation of DOPE into the nanoparticles significantly enhanced the in vitro transfection efficiency of pDNA-coated nanoparticles, as well as the resulting immune responses after different routes of administration [6,28–32]. 2.2. Deposition of cholesterol-mannan (chol-mannan) on the surface of the nanoparticles Human and mouse dendritic cells have been shown to express a mannose receptor [33,34]. Mannan, a 42 kDa Mw polysaccharide comprising 233 mannose residues, has been previously used to target DCs and enhance immune responses [35,36]. Cholesterol-modified mannan (chol-mannan) was used to anchor the mannan at the surface of the cured cationic nanoparticles. 1. Mix 1 mL of the pre-formed nanoparticle suspension (2 mg/mL) with 250 lg of chol-mannan, previously dissolved in hot water (5 mg/mL), and stir at room temperature overnight. 2. Alternatively, add chol-mannan directly to the warm oil-in-water microemulsion for similar results. This method eliminates overnight stirring. 2.3. Purification of nanoparticles by gel permeation chromatography 1. Soak Sephadex G-75 (powder) in de-ionized water for 24 h at room temperature prior to column preparation. 2. Remove free CTAB and chol-mannan by passing the nanoparticle suspension through a Sephadex G-75 column (14 65 mm) with water or 10% lactose as the mobile phase. Free CTAB can be easily removed from cationic nanoparticles by gel permeation chromatography (GPC) with no significant effect on cationic nanoparticle size. The loading volume of the nanoparticle suspension should be minimized to obtain better separation. 3. After GPC purification, pass nanoparticles through a 0.22 lm filter for sterilization.
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the surface of the nanoparticles before performing particle size and zeta potential measurements. 2.5. Particle size and zeta potential measurements of nanoparticles 2.5.1. Particle size 1. Prior to particle sizing, dilute the nanoparticle suspensions with water (0.2 lm filtered), so that the signal from the instrument, measured in counts per second (CPS), lies within the sensitivity range of 50,000 to 1 106 CPS. 2. Measure the particle sizes with an N4 Plus Sub-Micron Particle Sizer (Coulter, Miami, FL). Use plastic cuvettes (Beckman Coulter, Fullerton, CA; Cat. No. 7800091) at 25 °C for 120 s, scattering light at 90 °. As shown in Fig. 1, the particle size of mannancoated cationic nanoparticles was approximately 100 nm, whereas the coating of plasmid DNA resulted in about a 2.7-fold increase in particle size to about 270 nm. 2.5.2. Zeta potential Measure the zeta potential of the nanoparticles using a Zeta Sizer 2000 (Malvern Instruments, Southborough, MA). Mannan-coated cationic nanoparticles had a positive zeta potential of +41 mV. Upon coating with pDNA, the zeta potential decreased to +32 mV, as expected (Fig. 1). 2.6. Transmission electron microscopy We observed the size and morphology of nanoparticles cured from microemulsion precursors using a
2.4. Coating of plasmid DNA on the surface of the nanoparticles 1. Coat plasmid DNA (cytomegalovirus [CMV]-b-galactosidase) on the surface of the nanoparticles by gently mixing 1 mL of the purified and filter-sterilized nanoparticles in suspension with pDNA to obtain a final pDNA concentration of 50 lg/mL. 2. Let the formulation remain at least 30 min at room temperature for complete adsorption of pDNA to
Fig. 1. Particle size and zeta potential of mannan-coated cationic nanoparticles (Man-NPs) and pDNA-coated nanoparticles (Man-NPs with pDNA). The addition of pDNA to the mannan-coated nanoparticles caused an increase in the particle size (white bars) and a decrease in the zeta potential (filled circles), as expected.
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Fig. 2. Transmission electron micrographs. (A) Mannan-coated cationic nanoparticles and (B) pDNA-coated nanoparticles. Spherical and homogeneous cationic nanoparticles were engineered directly from the oil droplets of a clear microemulsion composed of emulsifying wax (2 mg), DOPE (100 lg), and CTAB (15 mM) in 1 mL water at 55 °C, cooling to room temperature during gentle stirring. Chol-mannan was added as described in Section 2. Mannan-coated cationic nanoparticles were purified by gel permeation chromatography, prior to the addition of pDNA. Plasmid DNA (CMV-b-galactosidase) was coated on the surface of the cationic nanoparticles (1 mL) to obtain a final pDNA concentration of 50 lg/mL.
Philips Tecnai 12 Transmission Electron Microscope in the Imaging Facility Unit, University of Kentucky. 1. On a carbon-coated 200-mesh copper specimen grid, glow-discharge the grid for 1.5 min. Deposit one drop of nanoparticle suspension on the grid and allow to stand for 1.5 min. Remove any excess fluid with filter paper. 2. Stain the grid with 1 drop of 1% uranyl acetate (0.2 lm filtered) and allow to dry for at least 30 min before examining under the electron microscope. As shown in Fig. 2A, the cationic nanoparticles were spherical and uniform, having an average particle size of 100 nm, which agreed with the results obtained by laser light scattering. Examination of the pDNA-coated nanoparticles showed surface pDNA as depicted by the darker staining pattern (Fig. 2B). 2.7. In vitro ConA agglutination assay Concanavalin A (ConA) is a plant lectin, a tetrameric protein with four binding sites specific for terminal mannosyl residues. Binding to the terminal a-D mannosyl residues of mannan will cause agglutination (or aggregation) of the complex in solution, resulting in an increase in turbidity. 1. Add increasing amounts of chol-mannan (standard) or mannan-coated nanoparticles (200 lL) at room temperature to 1 mL ConA (1 mg/mL) in 10 mM phosphate-buffered saline, pH 7.4, with 5 mM calcium chloride and 5 mM magnesium chloride. 2. Monitor the increase in turbidity at 360 nm for 200 s using a U-2000 Spectrophotometer (Hitachi Instruments; Danbury, CT). As shown in Fig. 3, mannan-coated nanoparticles with and without pDNA caused significant agglutination of ConA over 200 s, whereas both uncoated nanoparticles and free pDNA did not.
Fig. 3. Concanavalin A (ConA) agglutination assay to confirm the presence of mannan on the surface of mannan-coated cationic nanoparticles (Man-NPs) or pDNA-coated nanoparticles (Man-NPs with pDNA). Samples (0.2 mL) were added to 1 mL ConA (1 mg/mL) and the increase in turbidity at 360 nm was measured for 200 sec and compared with a ConA standard curve (insert). Control samples, either cationic nanoparticles (free NPs) or pDNA (free pDNA), did not cause ConA agglutination.
2.8. Immunization of mice We used 10- to 12-week-old female mice (Balb/C; Harlan Sprague–Dawley Laboratories, Indianapolis, IN) for all animal studies, observing NIH guidelines for the care and use of laboratory animals. 1. Anesthetize a mouse using pentobarbital (60 mg/kg body weight) by intraperitoneal injection. 2. On days 0, 7, and 14, immunize the mouse (n ¼ 5–6/ group) either with naked pDNA alone (5 lg), pDNA (5 lg)-coated nanoparticles, or b-galactosidase protein (10 lg) with the adjuvant Alum (Spectrum, 15 lg). 2.8.1. Topical immunization (derived from [29,32,37,38]) 1. Shave the hair on the back of the mouse with an A5 Single-Speed Clipper (Oster Professional Products,
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McMinnville, TN). Wipe the skin with an alcohol swab and allow to air dry. (One does not need to shave the hair for subcutaneous injections.) 2. Spread 100 lL of each formulation with a pipette tip onto the skin, covering an area of about 2 cm2 . Carefully disperse the solution over the skin without applying pressure.
2. Place the mouse on a 1-in.-thick foam pad, making sure the hind legs are moved from the abdomen underneath the mouse. Anesthetize by intraperitoneal pentobarbital injection as described above. 3. Position the Biojector 2000 device with a #2 intradermal spacer perpendicularly to the back of the mouse. Release the injectate (100 lL) carefully into the skin.
2.8.2. Subcutaneous immunization (derived from [28,31,32]) Briefly, pull the skin on the back of the neck upward to create a pinch of skin. Carefully insert the needle into the pinched skin and inject 100 lL of each formulation (in 10% lactose) with an insulin syringe with MICROFINE IV Needle (Becton–Dickinson, Franklin Lakes, NJ) on one site on the back, creating a bubble of liquid in the subcutaneous space.
2.9. Collection of biological samples
2.8.3. Jet injection immunization (derived from [6]) We used the Biojector 2000 needle-free jet injection device (Bioject, Portland, OR; Fig. 4), following the procedure for intradermal delivery in rats and mice. The Biojector 2000 delivers a titrated volume of liquid as a fine spray into the epidermis. The spray is driven by compressed carbon dioxide (CO2 ) gas contained in a small cylinder in the hand-held device (see www.bioject.com). 1. Shave a mouse (1–2 cm2 ) on the back area about halfway up starting at the base of the tail. Swab the shaved area thoroughly with 70% alcohol swab and allow to air dry.
2.9.1. Terminal blood collection 1. On day 28, anesthetize the mouse by intraperitoneal pentobarbital injection as described above. 2. For bleeding by cardiac puncture, tape the forelimbs of the mouse separately at a 90° angle to the body. Place a piece of tape across the lower abdomen of the mouse. 3. Insert a 21G 1 in. needle (with a 1 mL syringe) at an approximately 30° angle, immediately caudal to the xyphoid process. Aim the needle toward the left shoulder. Collect approximately 0.4–0.5 mL of blood. 4. Transfer the blood to a Vacutainer Brand Blood Collection Tube (Becton–Dickinson). Separate the serum centrifugation and store at )20 °C for analysis. 2.9.2. Spleen removal and preparation of splenocytes 1. Remove spleens from mice on day 28 and place in 5 mL HanksÕ Balanced Salt Solution (HBSS) (1) in a Stomacher Bag 400 (Fisher Scientific, Pittsburgh, PA). Homogenize the spleens at high speed for 60 s using a Stomacher Homogenizer.
Fig. 4. The Biojector 2000 (Bioject, Portland, OR) is a small handheld device that is used for needle-free delivery of antigen. It delivers a volume of liquid as a fine spray into the epidermis, the spray is driven by compressed carbon dioxide (CO2 ) gas contained in a small cylinder within the actual device itself.
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2. Transfer cell suspensions into 15 mL Falcon tubes and fill to 15 mL with 1 ACK buffer (156 mM NH4 Cl, 10 mM KHCO3 , and 100 lM EDTA) for red blood cell lysis. 3. After 5–8 min at room temperature, spin down the suspension 1500 rpm for 7 min at 4 °C. After pouring off the supernatant, re-suspend the cell pellet in 15 mL HBSS. Spin down the suspension at 1500 rpm for another 7 min at 4 °C. 4. After washing with 15 mL RPMI-1640 (BioWhittaker, Walkersville, MD), supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO) and 0.05 mg/mL gentamicin (Gibco-BRL, Gaithersburg, MD), re-suspend the cells in RPMI 1640 media (1 mL for each spleen in the pool). 2.10. Determination of antibody titers 1. To quantify b-galactosidase-specific serum antibody titers by ELISA, begin by coating Costar high binding 96-well assay plates with 50 lL b-galactosidase protein (8 lg/mL) overnight at 4 °C. Block the plates for 1 h at 37 °C with 4% bovine serum albumin (BSA)/ 4% NGS (Sigma) solution (100 lL/well) made in 1 PBS/Tween 20 (ScyTek Laboratories, Logan, UT). 2. Serially dilute mouse serum (50 lL/well) in 4% BSA/ 4% NGS/PBS/Tween 20 and incubate for 2 h at 37 °C. After washing three times with PBS/Tween 20 buffer, add anti-mouse IgG HRP F(ab0 )2 fragment from sheep (diluted 1:3000 in 1% BSA, 50 lL/well) and incubate for 1 h at 37 °C. Wash plates three times with PBS/Tween 20 buffer. Finally, develop the samples with 100 lL TMB substrate for 30 min at room temperature and stop with 50 lL of 0.2 M H2 SO4 . 3. Measure the OD of each well using a Universal Microplate Reader (Bio-Tek Instruments, Winooski, VT) at 450 nm. Assign the titer the highest dilution at which the OD450 is 2 SDs higher than that of the na€ıve group at equivalent dilutions. 4. Determine the antigen-specific serum IgA titer using a method similar to the IgG ELISA with modification. Specifically, serial dilute the serum samples at 1:5 in 4% BSA/4% NGS/PBS/Tween 20. In addition, peroxidase-linked goat anti-mouse IgA (1:6000 in 1% BSA) was used. Intradermal jet injection of pDNA-coated nanoparticles led to the highest antibody titer of all groups by any route with a geometric mean titer of over 160,000 (Fig. 5). The titer obtained with pDNA-coated nanoparticles delivered by jet injection was over 20-fold and 65-fold greater than that in mice immunized with pDNA-coated nanoparticles given by the subcutaneous and topical routes, respectively (p < 0:05). For naked pDNA alone, there was no significant difference in specific serum IgG titer (p ¼ 0:46) after jet injection and subcutaneous immunization, and both titers were sig-
Fig. 5. b-Galactosidase-specific IgG titer in the sera of mice at day 28. Mice were dosed on days 0, 7, and 14 with 5 lg pDNA in the form of naked pDNA (pDNA) or pDNA-coated nanoparticles (Man-NPs), or with b-galactosidase protein (10 lg) containing alum (15 lg) (bgal + alum). Data shown are the geometric mean titer with standard deviation (n ¼ 5–6) [6,28,29].
nificantly greater than after topical application of naked pDNA. By the subcutaneous route, the specific IgG titer in the sera of the mice immunized with pDNA-coated nanoparticles was 10-fold higher than that of the mice immunized with naked pDNA alone (p ¼ 0:039). After intradermal jet injection, the specific serum IgG titer was enhanced by more than 200-fold when mice were immunized with pDNA-coated nanoparticles, compared with jet injection with naked pDNA alone (p < 0:05). Immunization with pDNA-coated nanoparticles by jet injection led to more than a log increase in the IgA titer (mean of 800) elicited with naked pDNA given by either jet injection (p ¼ 0:047) or subcutaneous injection (p ¼ 0:072). Immunization with b-galactosidase protein formulated with Alum and administered by both jet and subcutaneous injection led to antigen-specific IgG and IgA titers, although the titers were statistically lower than those obtained by immunization with pDNAcoated nanoparticles. The dose of b-galactosidase protein and/or dosing regimen may have contributed, in part, to these low titers. 2.11. In vitro cytokine release and splenocyte proliferation We performed in vitro cytokine release assays and splenocyte proliferation assays using isolated splenocytes from immunized mice as described in previous reports [6,28–32]. By all routes of administration, pDNA-coated nanoparticles significantly enhanced Th1-type cytokine (IFN-c) production and splenocyte proliferative responses over naked pDNA alone. For example, immunization with pDNA-coated nanoparticles led to a
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5.6-fold enhancement in IFN-c production by jet injection (p ¼ 0:026) and a 2.8-fold enhancement by subcutaneous injection (p ¼ 0:006), compared with naked pDNA alone. Similarly, the pDNA-coated nanoparticles led to a 3.5-fold enhancement in IL-4 release by jet injection (p ¼ 0:03) and 2-fold enhancement by subcutaneous injection (p ¼ 0:07), compared with naked pDNA alone. Immunization with pDNA-coated nanoparticles resulted in a more balanced Th1/Th2 immune response by all routes of administration. Administration of naked pDNA alone did not have a significant effect on cytokine release from isolated splenocytes or in the proliferation of the splenocytes. 2.12. Statistical analyses We completed all statistical analyses using a one-way ANOVA, followed by pair-wise comparisons with FisherÕs protected least significant difference procedure, unless mentioned otherwise. A p value of 6 0.05 was considered to be statistically significant.
3. Conclusions Our research aims to rationally design genetic immunization methodologies based on nanoparticle delivery systems for plasmid DNA in combination with already available needle-free devices, such as the Biojector 2000. These methodologies may both reduce the dose of pDNA required and enhance the breadth and depth of protective immune responses (i.e., humoral and cellular). Methods have been developed to combine dendritic cell-targeted pDNA-coated nanoparticles with a needle-free device to significantly enhance antibody and Th1-type immune responses. The results from these studies demonstrated that a combination of improved pDNA delivery systems and an alternative route of administration significantly enhanced the breadth and depth of immune responses from a DNA vaccine.
Acknowledgments The authors thank Dr. Lawrence Baizer at Bioject Medical Technologies (Portland, OR) for his helpful comments and instruction on the use of the Biojector 2000.
Appendix. List of key materials Plasmid containing a CMV promoter with b-galactosidase reporter gene (CMV-b-gal) with endotoxin <0.1 EU/mg (Valentis, Woodlands, TX)
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Emulsifying wax (N.F. grade) (Spectrum Quality Products, New Brunswick, NJ) Aluminum hydroxide gel (Alum) (Spectrum Quality Products, New Brunswick, NJ) Hexadecyltrimethyl-ammonium bromide (CTAB) (Sigma Chemical, St. Louis, MO) b-Galactosidase protein (Sigma Chemical) Normal goat serum (NGS) (Sigma Chemical) Bovine serum albumin (BSA) (Sigma Chemical) Sephadex G-75 (Sigma Chemical) Phosphate-Buffered Saline/Tween 20 buffer (20) (ScyTek Laboratories, Logan, UT) Anti-mouse IgG, peroxidase-linked species-specific F(ab0 )2 fragment (from sheep) (Amersham–Pharmacia Biotech, Piscataway, NJ) Goat anti-mouse IgA, peroxidase-linked (Southern Biotechnology Associates, Birmingham, AL) Tetramethylbenzidine (TMB) (Pierce, Rockford, IL) Dioleoylphosphatidylethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL) {N-[2-(Chloesterylcarboxyamino)ethyl]carbamoylmethyl}mannan (chol-mannan) (Dojindo Molecular Technologies, Gaithersburg, MD) Mouse Interleukin-4 (IL-4) ELISA kit (Pierce-Endogen, Rockford, IL) Interferon-c (IFN-c) ELISA kit (Pierce-Endogen) CellTiter 96 Aqueous non-radioactive cell proliferation assay kit (Promega, Madison, WI) Concanavalin A (ConA) (Sigma Chemical)
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