**4. Conclusions**

DNA vaccines are still a promising option in the development of novel vaccination strategies. Although they have many advantages, the ability to induce effective immune responses in humans required for protection has been challenging. These challenges include ineffective delivery and poor uptake of DNA. Consequently, a recent focus has been in the development of delivery methods and/or inclusion of genetic adjuvants. Such genetic adjuvants are generally coexpressed with the antigen of interest or delivered through different plasmids. In the quest to develop and identify effective genetic adjuvants, a range of adjuvants was tested (HSP70, VSVG, IMX313, DTa, and PRF) and a novel and promising cytolytic DNA vaccine strategy has been developed. This cytolytic DNA vaccine is unique as it is based on a bicistronic plasmid with the ability to coexpress antigen and PRF in a balanced

mechanism causing necrosis of vaccine-transduced cells, followed by increased activation of immune cells and cross presentation of vaccine immunogen. Cytolytic DNA vaccines encoding nonstructural proteins of HCV have been tested to enhance immunogenicity of vaccine antigen in mice [57,107] and in a large preclinical animal model, the pig [56]. Likewise, increased immunogenicity and improved protection against EcoHIV challenge in mice with HIV Gag PRF [60] demonstrate the effectiveness of cytolytic DNA vaccines.

Adjuvants that provide effective costimulation for immune responses with specific immunogens may not have a similar effect with other immunogens, and therefore these need to be tested for their efficacy. Use of a genetic adjuvant such as PRF produces a suitable microenvironment for multiple/different immunogens and thus improves the delivery, immunogenicity and effectiveness of DNA vaccines. This strategy has considerable potential in the development of DNA-based vaccines against a range of infectious agents.

**Author Contributions:** Writing—concept and original draft preparation: A.C.S.; concept, review, and editing: B.G.-B., E.J.G., and D.K.W.; section contributions and review: M.G.M. and Z.A.M.

**Funding:** Cytolytic DNA vaccines developed in our laboratory and reviewed in this manuscript have been supported by the grants received from the National Health and Medical Research Council, Australian Centre for HIV and Hepatitis Virology Research, the National Foundation for Medical Research and Innovation, and The Hospital Research Foundation (THRF).

**Acknowledgments:** We thank THRF and the donor community for supporting the development of our novel DNA vaccines. Ashish C. Shrestha and Danushka K. Wijesundara are recipients of Early Career Fellowships from THRF.

**Conflicts of Interest:** The authors declare no conflict of interest.
