DNA Vaccine

DNA vaccination has become the fastest growing field in vaccine technology following reports at the beginning of the 1990s that plasmid DNA induces an immune response to the plasmid-encoded antigen after intramuscular injection into mice (Wolff et al. 1990). In theory, this conceptually safe, non-live vaccine approach is a unique and simple way to induce not only humoral but also cellular immunity. Whereas traditional vaccines rely on the production of antibodies through the injection of live attenuated virus, killed viral particles, or recombinant viral proteins, DNA vaccines are non-live, non-replicating, and non-spreading such that there is little risk of either reversion to a disease-causing form or secondary infection. In addition, DNA vaccines are highly flexible to encode viral or bacterial antigens, and immunological or biological proteins. DNA vaccines are stable, easily stored, and can be manufactured on a large scale. Furthermore, DNA vaccine avoids the risks associated to the manufacture of killed vaccine, as exemplified by the tainting of a polio vaccine with live polio virus owing to a production error (Offit et al. 2005). The earliest phase I clinical trial for a DNA vaccine was an HIV-1 candidate tested in individuals infected by HIV-1, followed by studies in volunteers who were not infected by HIV-1 (MacGregor et al. 1998). Subsequently, other therapeutic DNA vaccine trials against cancer, influenza, malaria, hepatitis B, and other HIV-1 candidates followed (Mincheff et al. 2000; Tacket et al. 1999; Le et al. 2000; Liu and Ulmer 2005; Ulmer et al. 2006). These trials demonstrated that the DNA vaccine platform is well tolerated and clinically safe. Some potential safety concerns including the integration into cellular DNA, the development of autoimmunity, and antibiotic resistance have been raised for DNA vaccines. DNA vaccines currently being tested do not show relevant levels of integration into host-cellular DNA (Kurth 1995; Manam et al. 2000; Ledwith et al. 2000; Temin 1990; Pal et al. 2006; Sheets et al. 2006). Furthermore, the preclinical or clinical evaluation of DNA vaccines have not indicated any insertional mutagenesis through the activation of oncogenes or the inactivation of tumor suppressor genes or any chromosomal instability through the induction of chromosomal breaks or rearrangements. To date, there has been no convincing evidence of anti-nuclear or anti-DNA autoimmunity developing in association with a DNA vaccine (MacGregor et al. 1998; Le et al. 2000; Klinman et al. 2000; MacGregor et al. 2000; Bagarazzi et al. 1997). As the antibiotic resistance genes contained by DNA vaccines are restricted to those antibiotics not commonly used to treat human infections, the risk of introducing antibiotic resistance into participants of clinical trials is very low. Alternative strategies to omit antibiotic selection in large-scale manufacture of DNA vaccines are being explored (Mairhofer et al. 2007; Cranenburgh et al. 2001). However, the first-generation DNA vaccines failed to demonstrate high levels of vaccine-specific immunity in large animals and humans.

On the basis of the notion that strong immune response can be elicited by the high level of antigen expression, significant efforts are ongoing to maximize gene expression and subsequent antigen expression from DNA vaccines. Firstly, for most recent DNA vaccines, the human CMV promoter is a common choice because it promotes high-level constitutive expression in a wide range of mammalian cells and does not suffer from downstream read-through (Donnelly et al. 1997). Alternatively, the use of muscle-specific promoters, such as promoters for creatine kinease or desmin, avoids constitutive expression of antigens in inappropriate tissues and also leads to the induction of antibody and T-cell responses, although levels of the antigen expressions are at least tenfold less than those driven by the CMV promoter (Bojak et al. 2002; Cazeaux et al. 2002). Secondly, many DNA vaccines use the bovine growth hormone terminator sequence or endogenous terminators to ensure proper transcriptional termination and efficient export of the mRNA from the nucleus (Montgomery et al. 1993). Thirdly, both enhancer elements and transcriptional transactivators, when placed either upstream or downstream of the open reading frame (ORF), can enhance promoter activity to further increase the expression of antigens (Barouch et al. 2005; Ito et al. 2003; Garg et al. 2004). Fourthly, "Kozak" consensus sequence (Kozak 1987; Kozak 1997) and codon optimization (Ikemura 1982; Ikemura 1985) are important to increase antigen production leading to enhanced T-cell and antibody responses by DNA vaccines (Garmory et al. 2003).

Additional efforts are ongoing to optimize the DNA vaccine platform in order to increase vaccine immunogenicity. Firstly, antigens can be targeted to the class I or class II processing pathways with the addition of sequences designed to direct intracellular trafficking. The immunogenicity of CD8+ T-cell epitope encoded by DNA vaccine was significantly enhanced when an adenovirus leader-sequence was fused in frame to target this CD8+ T-cell epitope to the endoplasmic reticulum (Ciernik et al. 1996). In some cases, the secretory signal leader sequence of tissue plasminogen activator (TPA) was fused with the N terminal of the antigens of interest to direct the antigens to the secretory pathway, which seemed to be another effective strategy to enhance the humoral immune response against the antigens (Li et al. 1999; Qiu et al. 2000; Ashok and Rangarajan 2002; Delogu et al. 2002). Secondly, antigenic proteins can be maximally truncated, leaving only defined epitopes for T-cells to induce immune response (Casares et al. 1997). This may be especially useful in cases where "full-length" proteins are toxic for the host (Barry and Johnston 1997) or immunosuppressive (Levy 1993). Thirdly, to overcome MHC restriction of individual epitopes or to induce a broader range of effector cells, multiple contiguous minimal epitopes in the form of a "polytope" can be delivered by DNA vaccine (Thomson et al. 1998). The immune potency of this strategy can be improved by including helper T-cell sequences to supply novel T-cell help (Stevenson et al. 2004). Fourthly, one current trend in DNA vaccine is the use of biodegradable polymeric microparticle-based and liposome-based delivery systems to induce cellular and humoral immunity (O'Hagan et al. 2006; Kutzler and Weiner 2008). Both polymer and liposome vehicles can protect DNA from degradation by serum proteins during transfer of DNA across membranes and after the release of generic material following fusion with endosomes. Lastly, multiple studies have shown that co-injections of DNA vaccine cocktails including plasmids encoding cytokines, chemokines, and/or costimulatory molecules along with antigens can have a substantial effect on the immune response to plasmid-encoded antigen (Leitner et al. 2000). Furthermore, synthetic oligodeoxynucleotides containing unmethy-lated CpG motifs can act as effective adjuvants that enhance both humoral and cellular immunity with minimal toxicity (Higgins et al. 2007).

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