**7. Conclusions**

It has been over 25 years since DNA vaccines were first introduced and many advances have been made in the field. However, despite showing promise in small animals, with some DNA vaccines being licensed for veterinary use [44,45], no DNA vaccines have been licensed for human use as immunogenicity is still relatively poor. Thus, a great deal of research has gone into improving the immunogenicity of DNA vaccines. Some of the strategies that have been shown to be effective are: (i) RNA optimization to remove mRNA structures that inhibit ribosomal loading and sequences that inhibit nuclear export of mRNA [46,47]; (ii) codon optimization [46,48,49]; (iii) use of Kozak sequences [50]; (iv) use of leader sequences to improve stability, translation, and secretion [46]; (v) use of 3 untranslated regions (UTR) such as polyadenylation signals and post-transcriptional response elements which are important for nuclear export, translation, and mRNA stability [51]; (vi) use of different promoters and enhancers [52–54]; (vii) the inclusion of genes expressing immunomodulatory molecules in the plasmid vector such as GM-CSF or IL-2 [55,56]; (viii) formulation of DNA vaccines in lipids and polymers [57]; (ix) use of better delivery systems [58–60]; and (x) use of suitable adjuvants [58,59].

In this review, we have only focused on a single method of improving DNA vaccine immunogenicity. This was the use of a short enhancer sequence derived from the circovirus PCV-1 capsid gene promoter to increase recombinant antigen expression. This enhancer element led to increased antigen expression and immunogenicity of HIV-1 subtype C candidate DNA vaccines and allowed for the use of 10-fold lower doses. The improved performance of the DNA vaccines with these candidates, compared to non-enhanced vectors that went into human clinical trial, has prompted the inclusion of the enhancer into all DNA vaccines under investigation in our research group, with excellent results. Future use of replicating circovirus-derived DNA expression and vaccine vectors may yet open up even more exciting possibilities.

**Author Contributions:** Both authors contributed equally to the review article.

**Funding:** This work is based upon research supported by the South African Medical Research Council with funds received from the South African Department of Science and Technology and the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation.

**Acknowledgments:** We acknowledge the pioneering work of Dr. Fiona Tanzer, and the gift of the original PCV-1 genome by Kenneth Palmer.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

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