598. Effective Humoral and Cellular Smallpox-Immune Responses Using a Combination of Plasmids Delivered by Intramuscular Injection Followed by Constant-Current Electroporation in Rabbits

Abstract

Previous studies have examined smallpox antigen-expressing plasmids in mice and non-human primates with limited efficacy. Recent studies showed that the electroporation technique can be used to dramatically increase plasmid uptake into the target tissues (e.g., skeletal muscle) when compared to simple injection. Consequently, the expression levels for the encoded transgene are orders of magnitude higher, likely improving potency. In a pilot experiment, uptake and distribution of plasmids with and without electroporation have been analyzed in the rabbit. Electroporated muscle showed a better distribution and uptake. We evaluated immune responses to smallpox antigens, after direct intramuscular plasmid injection followed by constant-current electroporation in groups of rabbits (n=3/group). The plasmids were administered either individually or in combination and their relative responses analyzed. Group 1 rabbits were immunized with a combination of eight plasmids expressing varying smallpox antigens (A13L, A14L, A27L, A33L, B5R, D8L, H3L, L1R). Group 2 rabbits were immunized with a combination of four different antigens (A27L, B5R, D8L, L1R). The final group received an immunization with an individual plasmid expressing a single antigen (B5R). All plasmids were administered via a single IM injection of 800 |[mu]|g (100 |[mu]|g each antigen and/or empty vector up to 800 |[mu]|g per injection) into semimembranosus muscle followed by electroporation using the ADViSYS constant-current EKD system, at 0.6 Amps, 3 pulses, 52 ms/pulse, 1 sec between pulses. Rabbits were immunized twice, at Study d 0 and at Study d 64. Sera were collected from rabbits at different time points, including on Day 85 and used to determine antibody responses by protein ELISA. Antibody responses were improved by electroporation in both immunized groups irrespective of the number of antigens delivered. A specific ELISpot assay for TNF is being developed to determine T-cell response rates in rabbits following DNA immunization. Rabbit PBMCs collected 2 weeks after first immunization were stimulated for 24h with 15mer peptides that span the entire length of the A27L gene. Immunization followed by electroporation induced improved antigen specific T-cell responses against A27L. Thus, the combination immunization strategy using smallpox antigens and constant-current electroporation appears important for driving better humoral and cellular-based responses in a relevant animal model.