Second Generation DNA Vaccines DNA vaccines have come a long way resulting in a second generation of vast improvements bringing a surge of interest to the community. In both small and large animal models, second-generation DNA vaccines show that they assist cellular and humoral immune responses. Additionally, research suggests that newer DNA vaccines can more broadly activate CD8+ cytotoxic T cells (CTL) in larger animal models, compared with earlier DNA methods. Inefficient uptake of plasmids by cells has been resulting in low immunogenicity. However, second generation DNA vaccine research has focused on developing novel strategies to enhance transfection efficiency and improve other facets of the DNA platform. These efforts include optimization of the antigens encoded by the plasmids to increase antigen expression on a per cell basis, improved formulation, and inclusion of molecular adjuvants to enhance and direct immune responses. Delivery Approaches Since the early 1990’s, the most common delivery for DNA vaccines has been intramuscular injection (IM). This method had a disadvantage of lowered specific immune responses. Therefore, several physical methods of delivery have been explored to increase the transfection efficiency of DNA vaccines, including needle-free approaches, such as particle bombardment and high-pressure mediated delivery, dermal patches, and electroporation (EP). Particle bombardment approaches use a highly pressurized stream to deliver vaccine plasmids on microscopic heavy metal beads. High-pressure mediated delivery is conceptually similar to particle bombardment. High-pressure delivery, the Biojector devices, deliver vaccines by forcing liquid through a tiny orifice to create a fine, high-pressure stream that penetrates the skin. Noninvasive dermal patch delivery utilizes a self-adhesive patch coated with multiple antigen or adjuvant encoding plasmids and a synthetic polymer that forms pathogen-like nanoparticles. Another promising physical method of delivery is EP, or the application of short electrical pulses to the delivery tissue, was initially studied over 25 years ago as a method to enhance the efficacy of chemotherapy agents. It was later discovered that EP also increases the uptake of DNA plasmids by cells, resulting in an increase in antigen production and in vaccine immunogenicity. Significant increases in immunogenicity have been observed while delivering DNA vaccines by intramuscular injection with electroporation, compared with IM alone. Thus, the use of improved delivery has enabled second-generation DNA vaccines to induce cellular immune responses comparable to viral vectors in nonhuman primates.