**1. Introduction**

Vaccines represent an effective strategy in the fight against infectious diseases and recent estimates suggest that vaccination prevents 2–3 million deaths every year [1]. The need for rapid and large scale vaccine production during epidemics against emerging pathogens is a major challenge in vaccine development [2], including effective vaccines for antigenically diverse and versatile pathogens that successfully subvert host immunity such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), and malaria [3–5].

DNA vaccines can overcome some of these challenges, as it is relatively easy to produce large number of doses within a short period of time, and they are stable at ambient temperature and do not require cold chain transportation. They are also consistent between lots and have an excellent safety profile allowing for safety evaluations by regulatory authorities and distribution in a large scale [6,7]. Importantly, DNA vaccines can induce both humoral and cell-mediated responses in the vaccinated host [8–10]. Although they are safe and well tolerated, they are often poorly immunogenic and inefficacious in humans [11]. Therefore, recent studies on the advancements of DNA vaccines are focused on effective delivery and increasing the immunogenicity of the encoded antigen/s of interest [12,13].

Effective immunization with DNA vaccines requires efficient transfection of host cells which is highly dependent on the delivery route and use of devices. Conventional delivery routes to introduce the DNA vaccine include intramuscular, intradermal, subcutaneous and oral routes [12]. The preferred delivery route depends on the requirement to activate specific immune cells. The skin is rich in immune cells including local dendritic cells (DCs) and natural killer (NK) cells, and is therefore likely to be a more favorable site for vaccine delivery [14,15]. Attempts to improve DNA delivery have been made through other physical methods with the use of 'gene guns' or electroporation, which transiently permeabilizes the cell membrane to efficiently transfer the DNA resulting in increased vaccine uptake by skin and muscle cells [16]. Although these methods have shown to increase DNA uptake [17,18], they require optimization to achieve increased efficiency and acceptance for clinical use. An alternative approach to improve transfection efficiency includes formulation of DNA with liposomes or nanoparticles [19]. Liposomal delivery can be affected by pre-systemic (epithelial) and systemic barriers (enzymatic degradation, binding, and opsonization) [20]. Encapsulation of DNA with nanoparticles has been reported to increase DNA uptake or transfection efficiency [21,22]. Some of the challenges in the use of nanoparticles with DNA include encapsulation inefficiency, endocytosis by target cells and toxicity [23].

The use of genetic adjuvants is one approach to enhance the immunogenicity of the antigen and can be used to complement other strategies (e.g., DNA delivery) also designed to improve the immunogenicity of DNA vaccines. Upon immunization with a DNA vaccine, the target cells uptake DNA by endocytosis [24] and the transfected cells express the DNA-encoded protein antigen(s). When antigen-presenting cells (APCs) are directly transfected, the intracellular proteins are processed and immunogenic epitopes are then presented by MHC Class I molecules, which can directly stimulate naïve CD8<sup>+</sup> T cells [12,25]. The protein immunogen released from transfected cells can be endocytosed and/or phagocytosed by other APCs and are presented by MHC class II molecules to activate naive CD4<sup>+</sup> T cells [25,26]. If the proteins are expressed by stromal cells like keratinocytes, APCs can also indirectly capture secreted antigens and cross-present by MHC Class I molecules to further stimulate CD8<sup>+</sup> T cells [27]. After DNA vaccination, CD8<sup>+</sup> T cells specific to the vaccine antigen undergo expansion, acquire effector functions and differentiates into memory CD8<sup>+</sup> T cells [28,29]. The memory cells differentiate into effector memory T cells upon re-exposure to the antigen [29,30]. The ability of a DNA vaccine to elicit T cell immunity is thus dependent on activating APCs to present antigen: MHC complexes to T cells [31] and adjuvants can serve as an important costimulatory factor to enhance this process.

In this brief review, based on our experience, we discuss the progress in the development of DNA vaccines, approaches to improve delivery and genetic adjuvants used to enhance immunogenicity. We focus on an innovative cytolytic DNA technology developed and patented in our laboratory.
