Total antigen-specific T cell responses were derived by combining the spot counts obtained with peptide pool I with the counts obtained with pool II. MAD-3 Statistical analysis Statistical comparisons were performed using a non-parametric Wilcoxon rank-sum test (SAS version 9.1, SAS Institute). use after thawing at room temperature. Finally, in a GLP-compliant repeat-dose toxicology study conducted in rabbits, single-vial Vaxfectin?-formulated vaccines, containing pDNA and Vaxfectin? up to 4.5 mg and 2 mg/injection, respectively, showed a favorable safety profile and were judged as well-tolerated. The results support further development of a Vaxfectin?-formulated pDNA vaccine to target congenital CMV infection. (TBCL) muscle using a 1cc tuberculin syringe fitted with a 21G 2 needle on Day 0. Identical vaccinations were performed on Day 21 in the left and on Day 49 in the right TBCL muscle. Approximately 80 sec after the vaccine was injected, muscles were electroporated using either a constant-voltage (MedPulser? DNA Delivery System, Inovio Biomedical Corporation) or a constant-current (ADViSYS electrokinetic device, EKD, ADViSYS, Inc.) device. Vaccine in the control group (no EP) was administered in the TBCL muscle of anaesthetized rabbits using similar 1cc tuberculin syringes fitted with a 21G 2 needle. With MedPulser?, two constant-voltage square electric pulses of 106 V of 60 msec duration each (nominal field strength 246 V/cm) were administered using 0.5 cm square gold plated four needle arrays (needle length 1.0 cm). With EKD, sterile 5-needle electrode arrays, in which the stainless steel electrodes were 1.0 cm apart in diameter, were used for EP. The guide disk of the array was adjusted so that the penetration depth of the electrodes was approximately 1.0 cm. After the array was inserted into the muscle, vaccine was administered through a central injection port located at the top of the array. The penetration depth of the injection needle was adjusted so that the bevel of the needle did not extend beyond the electrode array. The injection needle was removed, and the muscles were electroporated with three 0.6 A pulses (52 ms/pulse, 1 sec between pulses, constant-current pulse pattern #5).63,64 With both devices, a new electrode array was used for each rabbit muscle. Repeat-dose toxicology studies To assess the toxicity potential of SV Vaxfectin?-formulated vaccines, a good laboratory practices (GLP)-compliant repeat-dose toxicology study was conducted in New Zealand White rabbits (2.7C3.5 kg, n = 20 per group, evenly divided by sex). Rabbits received a bivalent vaccine (1:1 mass ratio of VCL-6365 and VCL-6368) formulated with Vaxfectin?, or PBS as a control, delivered as 1 mL unilateral IM injections with needle and syringe on Days 0, 21, and 42 (alternating limbs on subsequent injections). Two SV Vaxfectin? formulations 4-Hydroxyphenyl Carvedilol D5 were tested containing either 3 mg pDNA/2 mg Vaxfectin?, or 4.5 mg pDNA/1 mg Vaxfectin? (mg of total pDNA formulated with mg of total lipid, respectively). Animals were followed for up to 85 d and evaluated for clinical signs (including injection site reactogenicity), ophthalmology, body weight, food consumption, clinical pathology (hematology, coagulation and clinical chemistry), gross pathology (at necropsy), and histopathology as previously described.26 gB antibody ELISAs To detect serum gB-specific immunoglobulin G (IgG) antibodies, 4-Hydroxyphenyl Carvedilol D5 96-well plates were 4-Hydroxyphenyl Carvedilol D5 coated overnight at 2C8C with recombinant full-length human CMV gB protein purified from transfected Chinese hamster ovary cells (Austral Biologicals) at a concentration of 2 g/mL. Antibody levels, reported as endpoint titers, were determined as previously described.20 Serum gB antibodies were undetectable in all samples collected from rabbits and mice before vaccination (prebleeds) and tested at the starting dilution of 1 1:100. Unless otherwise stated in the text, gB-specific antibody responses were determined using ELISA plates coated with recombinant human CMV gB protein as described above. Antibody responses in some serum samples were analyzed using ELISA plates included in the CMV IgG Enzyme Immunoassay Test Kits (BioCheck, Inc.). These commercially available ELISA plates precoated with human CMV antigens were less sensitive than ELISA plates coated with recombinant gB protein, and they were only used to monitor temporal changes in antibody responses in rabbits immunized with VR-6365. IFN- ELISPOT assays Three weeks after the last immunization, T-cell responses to CMV antigens in vaccinated mice were determined by quantifying the number of splenocytes secreting IFN- in response to antigen-specific stimulation, as.