Increasing Gene Expression

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DCs have proven to be a notoriously difficult cell to transfect (59,90,112-115). Increasing transfection of these cells seems to be a logical way to increase vaccine potency. Although some evidence suggests that greater antigen expression does not always lead to greater immune responses (116), others have shown that increasing the magnitude and duration of antigen expression is a viable way to increase the immuno-genicity of genetic vaccines. Some examples of these strategies are: (1) optimization of the plasmid construct, (2) avoiding degradation in the lysosomes, (3) increased DC lifespan, and (4) self-replicating antigen constructs.

4.4.1. Plasmid Modifications

One of the most straightforward ways to increase gene expression is through the addition of multiple gene expression cassettes in the same plasmid vector. Sasaki et al. used these dual-antigen expression vectors to generate significantly higher expression than that obtained by using 2x the amount of single expression vector cassettes. Vaccinations with these plasmids correspondingly led to increased IL-4 and IFN-y secretion by isolated splenocytes (117). Haas et al. demonstrated that optimizing codon usage, which can be significantly different in mammals relative to bacteria, led to increases in antibody and CTL responses in mice using a HIV gp120 antigen construct (118). Another example is codon optimized plasmid encoding for a MHC class I restricted listeria antigen, which showed increases in CTL responses and partial protection from listerial challenge while unoptimized plasmid remained ineffective (119).

4.4.2. Avoiding Lysosomal Degradation of Plasmid

Other attempts to increase gene expression are aimed at avoiding lysosomal degradation of the plasmid DNA. Trehalose 6,6'-dimycolate (TDM) has been shown to cause inhibition of fusion between the lysosome and phagosome (120) and this inhibition may allow more time for the transfer of DNA from phagosomal compartments to cytoplasm of APCs before lysosomal degradation. Inclusion of TDM in PLGA micropar-ticle vaccine formulations induces stronger resistance to mycobacterium tuberculosis in mice (121). Other strategies attempt to avoid the lysosomal pathway altogether by adding mechanisms for traversing the plasma membrane (122). Using a plasmid encoding either the protein transduction domains for HSV-1 (VP-22) (123), or Pseudomonas aeruginosa exotoxin A (ETA(dll)) (124), fused with HPV type 16 E7 antigen, Hung et al. observed a 50x increase in the amount of responding CD8+ T-cells along with the increased ability of vaccinated mice to react to E7 expressing tumors.

4.4.3. Dendritic Cell Life Span

Increasing the lifetime of an antigen expressing DCs in vivo is yet another strategy to increase the immune presentation. Kim et al. investigated the effect of including a plasmid encoding antiapoptotic proteins such as Bcl-xL (125) and serine protease inhibitor 6 (SPI-6) (126) to antigen fusion constructs with MHC class II targeting signals. These antiapoptotic proteins increased avidity of T-cells and elicited stronger tumor protection. Interestingly, covaccination with genes such as Fas (127) and cas-pases 2 or 3 (128) (apoptotic proteins) can also increase the potency of genetic vaccine formulations. While the exact mechanism of immune stimulation is unclear, it is possible that cross presentation of antigen from the apoptotic cells to a DCs may serve as an appropriate "danger" signal.

4.4.4. Replicons

Self-replicating RNA antigen constructs, or replicons, are based on alpha viruses such as the Venezuelan equine encephalitis virus, Sindbis Virus, and Semliki Forest Virus. Plasmid replicons contain the information for the transcription of a positive strand of RNA, which in turn encodes for both a 5' replicase complex, and a negative strand of antigen encoding RNA (see Fig. 7). These vectors do not produce viral structural proteins, leaving no possibility for recombinant events. This is accomplished by replacing the viral gene for the structural proteins with a heterologous gene. Replicons have also been called "suicide vectors" because the presence of large quantities of dsRNA is thought to induce apoptosis shortly after transfection. Because of the infection process occurring in the cytoplasm, there is little possibility of chromosomal integration.

It should be noted that by using a defective helper gene encoding structural proteins, an infection competent, but replication incompetent, viral particle can be produced. These particles can target DCs (129,130) and have higher gene transfection efficiency than replicon plasmids alone. However, there is a small probability that recombination events could occur, leading to infectious particles. The reader is directed to a recent review on alphaviral vectors for more detail on this topic (131).

Replicons have proven to be powerful enhancements to DNA vaccination, and are capable of eliciting antibody and tumor protective responses at up to 1000 times lower titers than conventional naked DNA vaccines in a P-gal expressing tumor model (132). Vaccination with replicons has also induced protective immunity to melanoma challenge in a TRP-1 expression system, unlike conventional DNA vaccines (133). Although it is

Fig. 7. Self-replicating plasmid replicons.

logical to infer that increased antigen expression is the reason for this enhancement, it is widely accepted that is rather results from the presence of dsRNA. The formation of dsRNA can activate antiviral apoptosis pathways, which subsequently lead to cross-priming of antigen in the presence of a danger signal (133).

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