On the use of DNA vaccines for the prophylaxis of mycobacterial diseases.

The principle of genetic vaccination using naked bacterial plasmid DNA is surprisingly simple, yet the first reports on this technology were only made about a decade ago. In their seminal paper of 1990, Wolff et al. reported that injection of naked plasmid DNA encoding the bacterial enzyme -galactosidase into muscle cells could lead to direct gene transfer to the muscle cells, transcription in the nuclei, and subsequent synthesis of the enzyme (77). In 1992, Tang et al. demonstrated that plasmid injection could be used to elicit an immune response (60), and the next year, Ulmer et al. were the first to report on the protective efficacy of DNA vaccination against an infectious disease, i.e., influenza A (71). Since then, DNA vaccines have been reported to induce protective immunity in numerous animal models of parasitic, viral, and bacterial diseases (14, 25). DNA vaccines have a number of potential advantages, including ease of preparation, stability, relatively low cost, and safety for immunocompromised patients. A number of clinical trials are actually being performed in the fields of cancer, human immunodeficiency virus (HIV), and malaria. In a DNA vaccine, the gene for an antigen is inserted into a bacterial plasmid vector, plasmid DNA is amplified in transformed bacteria, and the purified plasmid DNA encoding the immunogen is injected into an immunocompetent host. Cellular targets are, first of all, muscle cells (intramuscular immunization) and keratinocytes (epidermal gene gun immunization), but bacterial DNA also gets into professional antigenpresenting cells (APCs) present in and attracted to the injection site. Once inside the cell, the plasmid translocates to the nucleus in a way that is not really understood. Transcription of the coding information is driven by a strong viral or eucaryotic promoter, very frequently the promoter of IE1, the first immediate-early antigen of cytomegalovirus, followed by its first intron (intron A). The gene of interest is followed by a eucaryotic transcription-termination polyadenylation site, which enables efficient translation and termination of the protein by the ribosomes. An origin of replication (OriC) and an antibiotic (kanamycin or ampicillin) resistance marker are used for selective amplification of the plasmid in Escherichia coli. The methylation pattern of injected plasmid DNA remains of a bacterial nature (methylated adenosines) up to 19 months after injection, indicating that, indeed, no replication occurs in the eucaryotic host (76). Furthermore, sequence homology with eucaryotic sequences is minimal and as a result of these two factors, the risk for homologous recombination and mutagenic or potentially carcinogenic integration is very low. Random integration may take place but at chances of 1 copy in 150,000 nuclei (48).