Table 1.

Optimization strategies for DNA vaccines

Categories	Approaches	Effects		Safety	Practical considerations
		Pros	Cons
Codon optimization	Use of host codons to maximize antigen expression	Most studies have shown increased antigenicity and immune responses (Cheung et al., 2004; Lin et al., 2006; Megati et al., 2008; Ngumbela et al., 2008; Siegismund et al., 2009; Tenbusch et al., 2010; Ternette et al., 2007; Trollet et al., 2009; Zhu et al., 2010). (These studies were carried out in mice)	In some cases no enhancement was observed or codon optimized construct was inferior to native sequence (Dobano et al., 2009; Varaldo et al., 2006). (These studies were carried out in mice)	No safety concerns	When antigen expression level is a problem, codon optimization should be considered
Promoter optimization	Use of strong viral promoters (CMV, SV40).	High expression and antigenicity in most cases (Chapman et al., 1991, monkey cells; Cheng et al., 1993, mouse, rabbit and rhesus monkey; Manthorpe et al., 1993, mouse; Wang et al., 2006, mouse).	Some promoters may have suppressive effects on antigenicity or may be inhibited by endogenous cytokines (Cao et al., 2011, mouse; Gribaudo et al., 1993, mouse fibroblast cells; Kerr and Stark, 1992; Vanniasinkam et al., 2006, mouse; Xiang and Ertl, 1995, mouse).	No safety concerns	Strong viral promoters are the first choice to achieve high antigen expression
	Inducible or endogenous promoters.	Showed greater efficacy for specific DNA vaccines (Cao et al., 2011, mouse; Vanniasinkam et al., 2006, mouse).	Not broadly effective.
Reduction of bacterial elements	Delete redundant bacterial sequences	e.g. pcDNA3.1 is upgraded to pVAX1	In some cases, may be time consuming.	Increased safety	More collaboration is needed to standardize new technologies and achieve consistent evaluation.
	Sucrose induction system	Replace the bacterial selection marker (Luke et al., 2009, mouse; Luke et al., 2011b, rabbit).
	Mini-circle technology	Maintain the minimum antigen expressing cassette (Jechlinger et al., 2004; Kay et al., 2010, mcDNA preparation technology; Narsinh et al., 2011, stem cells; Osborn et al., 2011, mouse; Zuo et al., 2011, cultured cancer cells and mouse).
S/MAR vectors	Generation of episomal vectors	Decrease the integration rate and increase long-term expression (Argyros et al., 2011, cultured cells and mouse; Conese et al., 2004, review).	No commercial standard. Need more evaluation.	Increased safety	This method can be combined with mini-circle technology to achieve maximum safety and efficiency
Targeting technologies	Targeting to APCs (ligand fusion and other methods)	Enhanced antigen processing and presentation (Anwar et al., 2005; Argilaguet et al., 2011; Boyle et al., 1998; de Arruda et al., 2004; Faham et al., 2011; Ji et al., 1999; Liu et al., 2011; Lu et al., 2003; Marques et al., 2003; Midha and Bhatnagar, 2009; Niazi et al., 2007; Palumbo et al., 2011; Rigato et al., 2010; Wang et al., 2011b; Yang et al., 2009). (These studies were carried out in mice)	Some studies showed no enhanced effects (Carvalho et al., 2010, cultured cells; Kaur et al., 2009, mouse; Vidalin et al., 1999, mouse).	Potential for interference with the endogenous immune system needs to be evaluated	Targeting effects need to be tested individually. More suitable for anti-cancer DNA vaccines
	Subcellular targeting (use endogenous trafficking system)
Adjuvants	Genetic adjuvants, e.g. cytokines and chemokines	Enhanced immune responses were observed in many studies (Bode et al., 2011, review; Kim et al., 2008, mouse; Kolka et al., 2005, mouse; Liu, 2011, review; Luke et al., 2011a, mouse; Riedl et al., 2006, cultured cells and mouse; Schirmbeck et al., 2002, mouse; Tovey and Lallemand, 2010, review).	Co-expression of inflammatory cytokines or chemokines has the potential for increased side effects	Potential interference with the endogenous immune system needs to be evaluated.	Considering uncertain safety issues may be more suitable for use in therapeutic DNA vaccines, e.g. cancer vaccines
	Modulation of the pattern recognition receptors, e.g. TLRs, RLRs and NLRs
	Other adjuvants
Epigenetics	Drugs regulating DNA methylation or histone modifications	Some initial promising potential for DNA vaccines (Brooks et al., 2004, rat; Fujisawa et al., 2011, mouse model; Gowda et al., 2011, mouse; Karpf, 2006, review; Pearce and Shen, 2006, review; Riu et al., 2007, mouse; Xu et al., 2011, mouse; Youngblood et al., 2010, review).	Both mechanistic and applied studies are in early stages	Safety issues not known	Need extensive studies, which will provide basis for designing new DNA vaccine strategies.
	Avoiding epigenetic silencing
	Epigenetics mechanisms of immune responses
RNAi technology	Targeting potential suppressive factors.	Enhanced immune responses were observed in some studies (Geiben-Lynn et al., 2011, mouse; Greenland et al., 2007, mouse; Huang et al., 2008, mouse; Kim et al., 2011, mouse; Kim et al., 2005, mouse; Wang et al., 2011c, mouse).	More studies are needed to identify the appropriate factors to be targeted.	No safety issues have been identified to date but still early days	This technology will serve as a powerful tool when appropriate targets are identified.
Systems or “omics” technology	Systems biology	These studies provide valuable guides for DNA vaccine design (Nakaya et al., 2011, human, mouse model; Rappuoli and Aderem, 2011, review; Sette and Rappuoli, 2010, review; Trautmann and Sekaly, 2011, review; Wang et al., 2011d (human and mouse; Xiao et al., 2011, mouse; Yang et al., 2006, in silico and arry study).	Requirement of advanced technology and expensive experiments currently limit use to large companies	No safety issues	These technologies will greatly enhance the rational design of future DNA vaccines.
	Library technology
	Proteomics
	Genomics (reverse vaccinology)
	Other “omics” and combinations