The utility of plasmid DNA as an immunogen has been limited by its weak immunogenicity. In the present study, we evaluated the ability of a family of linear polyethylenimine (PEI) polymers, complexed to plasmid DNA, to augment DNA expression in vivo and to enhance antigen-specific adaptive immune responses. We showed that four of five structurally different PEIs that we evaluated increased in vivo DNA expression 20-to 400-fold, and enhanced DNA-induced epitope-specific CD8 IntroductionDNA vaccine constructs have been evaluated and are being tested in a number of human clinical trials for generating immunity to many types of diseases, including infectious, cancer, allergic, and autoimmune diseases [1,2]. In addition, the use of DNA vaccinesCorrespondence: Dr. Evita V. Grant e-mail: evita.grant@gmail.com has proven effective in some current veterinary products such as a vaccine against infectious hematopoietic necrosis virus for salmon [3], melanoma immunotherapeutic vaccine for dogs [4] and a vaccine against the West Nile Virus for horses [5]. Unfortunately, the immunogenicity of plasmid DNA in humans has proven to be modest in comparison with the immunogenicity observed in other species of microbial expression vectors. Consequently, efforts are being made to increase the immunogenicity of DNA vaccine constructs. Strategies have been developed to increase plasmid DNA expression via codon modification [6], the use of enhanced promoters [7] and the use of microbial transcriptional C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 2938 Evita V. Grant et al. Eur. J. Immunol. 2012. 42: 2937-2948 control elements [8]. Since plasmid DNA has poor in vivo transfection efficiency, a number of formulations of plasmid DNA have been evaluated to protect DNA from degradation and to enhance transfection efficiency [9][10][11]. The "gene gun" technology [12] was developed to enhance DNA delivery and immunogenicity. Furthermore, immunomodulation strategies have been evaluated for their ability to enhance DNA immunogenicity [13][14][15]. The synthetic polycationic polymer polyethylenimine (PEI), consisting of chains of ethylenimine units-CH 2 CH 2 NH-, has become the gold standard for enhancing the in vivo expression of administered genes [16,17]. PEI is superior to other nonmicrobial transfection agents because of its ability to protect DNA from degradation [18], to escape intracellular endosomal lysis [19] and to efficiently deliver DNA into the nucleus of cells [20]. Formulation with PEI polymers has been employed in DNA-and siRNAbased immunotherapy against cancer [21,22] and in vaccination studies targeting different infectious agents [10,23,24].In the present study, we have evaluated the physicochemical characteristics of a series of PEI polymers and have assessed associations between particular characteristics of these polymers and their ability to facilitate in vivo DNA expression and enhanced adaptive immune responses. Moreover, we have examined plasmid DNA constructs formulated with these various PEI...