Plasmids
A plasmid encoding the HBSAg under the control of the Human Cytomegalovirus Immediate Early gene I (HCMVIE1) enhancer/promoter (pRc/CMV-HBs(S) was obtained from Aldevron (Fargo, ND) [20]. A plasmid encoding the firefly luciferase gene under the control of the HCMVIE1 enhancer/promoter (pHCMVIE1-luciferase) was used to measure relative gene expression levels following gene transfer by low-volume jet injection, particle bombardment and needle and syringe injection [2]. Plasmids were propagated in DH5α cells and purified by Bio 101 Merlin Core Services (Carlsbad, CA).
Animals, skin preparation, and injection schedules
Twenty-five male New Zealand White rabbits were obtained at 2–3 kg in weight. Animals were purchased from a commercial vendor (Covance, Denver, PA). Health status was evaluated quarterly by the vendor and the animals were free of tested pathogens (other than Bordetella bronchiseptica and rotavirus but these were not expected to interfere with the current study). The rabbits were in excellent health and not symptomatic. The rabbits were anesthetized with 40 mg/kg Ketamine (100 mg/ml,) and 3 mg/kg Xylazine (20 mg/ml) prior to each injection. In preparation for injection, the dorsal skin was shaved, treated with a depilatory, and washed with a chlorhexidine scrub followed by 70% alcohol. Following injections 0.05 mg/kg Buprenorphine (0.3 mg/ml) was given for analgesia.
To establish skin penetration depth following low-volume jet injection, one rabbit was injected at 6 different sites with a 1% India ink solution. The skin was removed at necropsy and examined under a dissecting microscope to measure the length of the injection paths. To establish optimal injection conditions for low-volume jet injection, two rabbits were injected at a total of 15 sites per rabbit with pHCMVIE1-luciferase and euthanized 48 h later. The injected skin was removed at necropsy and tested for luciferase activity. When using particle bombardment the goal is to deliver the majority of gold particles into the epithelial cell layer rather than the deeper, less cellular dermis [21]. To establish skin penetration depth following particle bombardment, four rabbits were injected at eight sites on each rabbit. The skin was removed at necropsy and fixed in 10% neutral formalin, embedded by standard methods, and 5-micron sections were prepared. Hematoxylin and eosin stained sections were examined under the microscope to determine the penetration depth. Gold particles were found distributed primarily in the epidermis and minimally in the superficial dermis of the rabbit skin (data not shown). To establish optimal injection conditions for particle bombardment, six rabbits were injected at a total of 12 sites per rabbit with pHCMVIE1-luciferase and euthanized 48 h later. The injected skin was removed at necropsy and tested for luciferase activity. To establish optimal conditions for intra-dermal needle and syringe injection, two rabbits were injected at a total of 7 sites per rabbit with pHCMVIE1-luciferase and euthanized 48 h later. The injected skin was removed at necropsy and tested for luciferase activity.
For the immunization experiments, rabbits were injected at three time points at two weeks intervals: 0 days, 14 days and 28 days. Three ml blood was obtained from an ear vein prior to each injection and two weeks and four weeks following the third and final injection. One of the five rabbits that received particle bombardment injection died under anesthesia prior to the second injection. A necropsy was performed, no abnormalities were found, and the death was attributed to anesthesia.
Optimization of low-volume jet injection, particle bombardment and needle and syringe injection conditions
Low-volume jet injection was performed with a prototype low-volume jet injector with adjustable injection volume and pressure. The plasmid DNA solution at 1 μg/μl was prepared in phosphate buffered saline (PBS). The injector is manufactured with a refillable liquid reservoir that holds 180 μl and can rapidly deliver multiple injections to the same site (EMS, Konstanz, Germany) [2, 22]. For optimization of the jet injection experiments, three different injection volume settings (5-μl, 6-μl and 7-μl) at three different pressures (1,2 and 3-Bar) were tested. When using low-volume jet injection for intradermal delivery the goal is contain the delivery solution within the epidermis and dermis but optimize gene expression levels. Conditions for optimal gene expression were established by testing luciferase activity in the skin 48 h following injection of pHCMVIE1-luciferase. Four rapid injections at each volume were used for total DNA delivery of 20 μl /20 μg plasmid DNA, 24 μl/24 μg plasmid DNA, and 28 μl/28 μg plasmid DNA. The injected fluid remained within the dermis under all conditions. The delivery volume with the highest mean luciferase expression was chosen as the optimal delivery condition for the immunization studies. This optimal setting of 28 μl/28 μg plasmid DNA delivered at 3-Bar was utilized for each immunization time point.
A Helios Gene Gun System, (Bio-Rad Laboratories, Hercules, CA) was utilized for particle bombardment. Plasmid DNA was precipitated onto gold particles 1.6 microns in diameter following standard protocols [21]. Optimization conditions tested included the helium pressure, concentration of polyvinylpyrrolidone (PVP), amount of carrier gold and DNA concentration as described previously [23]. Conditions for optimal gene expression were established by testing luciferase activity in the skin 48 h following injection of pHCMVIE1-luciferase. The set of conditions (pressure/PVP concentration/gold and DNA amounts) with the highest mean luciferase expression and optimal skin penetration was chosen as the optimal combination for immunization studies. This optimal setting of one μg plasmid DNA prepared with 0.05 mg/ml PVP/0.50 mg gold delivered at 360 psi was utilized for each immunization time point.
A plasmid DNA solution at 1 μg/μl was prepared in PBS. A 29-gauge needle and 1 ml syringe were utilized for intra-dermal injection. Conditions for optimal gene expression in the injected skin were established prior to the genetic immunization experiments. When using needle and syringe injection for intradermal delivery the goal is contain the delivery solution within the epidermis and dermis but optimize gene expression levels. Conditions for optimal gene expression were established by testing luciferase activity in the skin 48 h following injection of pHCMVIE1-luciferase. A maximum of 20 μl/20 μg plasmid DNA could be injected and kept contained within the epidermis and dermis. This volume yielded the highest luciferase values. The optimal condition of twenty μl (20 μg plasmid DNA) was delivered at each immunization time point.
Luciferase Assay
Two days after injection rabbits were euthanized and the area of injected skin excised. Skin tissue was homogenized in lysis reagent buffer (Luciferase Assay System, Promega, Madison, WI). For each injection sample, luciferase activity was measured in duplicate using a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA) [2].
Enzyme-linked immunosorbent assay (ELISA)
Affinity-purified recombinant Hepatitis B antigen produced in Saccharomyces cerevisae following transformation with the plasmid pCGA7 was used for detection of antibody responses using an ELISA (Aldevron, Fargo, ND) [20]. The recombinant antigens were diluted in 0.1 M carbonate buffer (pH 9.6) and coated at 1 ug/100 ul per well in 96-well microtitre plates (Dynex Technologies, INC. Chantilly, VA). Adjoining negative control wells were coated with 0.1 M carbonate buffer (pH 9.6) alone. The plates were incubated at 4°C overnight. Plates were washed three times in phosphate buffered saline (PBS) (pH 7.4) and blocked with 5% bovine serum albumin (BSA) in PBS before serum was added. One hundred ul of PBS-Tween-20 with 1% BSA was added to each well. To quantify the antibody response at the different time points after injection(s) for each rabbit, 100 ul of diluted serum samples at 1:25, 1:250 and 1:2,500 were added to separate wells in duplicate. Assays for all time points were performed simultaneously. The entire assay was repeated at least twice for all samples. Rabbit sera obtained prior to any injection were utilized as negative controls. The plate was incubated at 37°C for 1 h. After additional washing, phosphatase-conjugated goat anti-rabbit IgG antibody was added into each sample well and incubated for 1 h at 37°C. Diethanolamine (DEA) buffer phosphatase substrate (Kirkegaard Perry Lab, Gaithersburg, Maryland) was added into all wells and the plates were incubated for 30 min at room temperature in the dark. The reaction was terminated after the appearance of color using 3 N NaOH. The absorbance was measured at 410 nm using a microplate reader (Emax, Sunnyvale, CA). The mean value for each set of duplicate wells was determined. Specific binding to the Hepatitis B antigen was determined by subtracting the mean value from the negative control wells that were coated with buffer alone from the mean value from the Hepatitis B-coated experimental wells. Rabbits defined as demonstrating an immune response exhibited a statistically significantly higher mean absorbance reading following injection measured at serum dilutions of 1:250 and 1:2,500 at a minimum of two serial time points following injection as compared to mean values for negative control serum.
Statistical analysis
Means and standard errors (S.E.) for the luciferase expression assays and antibody responses as measured by ELISA were calculated (SPSS 9.0, SPSS, Inc., Chicago, IL). Mean antibody responses were compared by ANOVA (SPSS 9.0). P ≤ 0.05 was considered a statistically significant difference.