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tion strategies, such as whole killed or live attenuated virus and recombinant protein-based vaccines, without specific shortcomings and inherent risks associated these vaccination methods. Like inactivated or subunit vaccines, DNA vaccines appear conceptually safe because they are nonreplicating and nonlive. In contrast to inactivated or subunit vaccine, DNA vaccine cassettes produce immunological responses that are more similar to live vaccine preparations. By directly introducing DNA into the host cell, the host cell is essentially directed to produce the antigenic protein, mimicking viral replication or tumor cell marker presentation in the host. Unlike a live attenuated vaccine, conceptually there is little risk from reversion to a disease-causing pathogen from the injected DNA, and there is no risk for secondary infection as the material injected is not living and not infectious. Furthermore, mul-ticomponent DNA vaccines can be engineered to include specific immunogens, which could optimize and amplify desirable immunological responses. Perhaps this ability to target multiple antigenic components may be a particularly important characteristic of DNA vaccines because multicompo-nent DNA vaccines can be engineered to include specific immunogens that could optimize and amplify desirable immu-nological responses.

D. Mechanism of DNA Vaccines

The exact mechanism for DNA immunization has been a center of major debate (11-13), but is likely to be similar to traditional antigen presentation. In the body's immune system, cells need to process and present antigenic peptides to lymphocytes in order to stimulate antigen-specific immune response. Thus, antigen must be processed and presented to T lymphocytes by antigen-presenting cells (APCs) (14). Antigen presentation and recognition is a complex biological process that involves many interactions between antigen-presenting cells and T cells (Fig. 3). There are 4 primary components that are critical in the professional APC's ability to present the antigen to T cells and activate them for appropriate immune responses. These components are major his to compatibility complex [MHC]-antigen complexes, costimulatory molecules (primarily CD80 and CD86), intracellular adhesion molecules, and soluble cytokines. Naive T cells circulate through the body across lymph nodes and secondary lymphoid organs such as the spleen. Their migration is mediated among other factors by intercellular adhesion molecules and cytokines. As the T cells travel, they bind to and dissociate from various APCs. This action is mediated through adhesion molecules. When a naive T cell binds to an APC-expressing relevant MHC/peptide complex, the T cell expresses high levels of high-affinity IL-2 receptor. Only when this T cell receives a costimulatory signal through CD80/CD86-CD28 interaction does the T cell make soluble IL-2, which then binds to the receptors and drives the now-armed effector T cell to activate and proliferate.

Antigen is expressed at significant levels in muscle, following intramuscular inoculation of plasmid DNA (6). Using reporter gene injections in mice, various investigators have reported the detection of gene expression after intramuscular (IM) injection of DNA expression cassettes (6). Protein expression was detected in the quadriceps muscle of mice after injecting plasmid vectors encoding chloramphenicol ace-tyltransferase, luciferase, and p-galactosidase reporter genes into the muscle.

Muscle cells have several structural and functional features that seem to make them well suited for DNA uptake in vivo (15). Muscle consists of multinucleated contractile muscle fibers with cylindrical shape and tapered ends. These muscle fibers have mycogenic stem cells attached to them. When the muscle fibers are damaged or stressed, the stem cells are activated. The resulting myoblasts proliferate and eventually fuse to form new muscle fibers. It is believed that this continual activation and proliferation of the myoblasts allow a more opportunistic uptake of injected DNA. Because it has been shown that the uptake of the injected DNA and the subsequent production of protein occurs in muscle cells, they have been proposed as a potential site of antigen processing andpresenta-tion. However, the myocytes, which make up the muscle tissues, do not express CD80 or CD86 costimulatory molecules needed for efficient presentation, although a new study has identified an additional costimulatory molecule distinct from CD80 or CD86, which can be expressed in muscle cells (16). The question of ability of muscle cells to provide costimula-tory signals drives the current debate in the literature about the mechanism of antigen presentation following intramuscular DNA immunization.

Figure 3 Effective T cell activation by APC. The interaction between antigen-MHC and T cell receptor leads to the expression of IL-2 receptor. This T cell proliferates when the second signal is provided from APC's costimulatory proteins. CD28-CD80/CD86 ligation initiates the production of IL-2 production and leads to the proliferation of activated T cell.

Figure 3 Effective T cell activation by APC. The interaction between antigen-MHC and T cell receptor leads to the expression of IL-2 receptor. This T cell proliferates when the second signal is provided from APC's costimulatory proteins. CD28-CD80/CD86 ligation initiates the production of IL-2 production and leads to the proliferation of activated T cell.

One potential mechanism is that the antigens produced in muscle are secreted from transfected muscle cells or released due to cell apoptosis (11,12). Such exogenous antigen could then be taken up by professional APCs in the draining lymph nodes, where the antigen is processed via the MHC class I pathway of these cells. Then, these APCs are hypothesized to present the processed peptides to T cells. Recently, there have been reports that indicate that immune system has an inherent mechanism by which exogenous antigens access MHC class I molecules. One recent report identified dendritic cells as the potent mediator of such presentation antigen derived from phagocytosed apoptotic cells (17). Immature dendritic cells engulf apoptotic cells and cross-present antigen from these sources to induce class I-restricted CTLs.

Another possible mechanism is the direct transfection of professional APCs by the injected DNA. Such a mechanism may be more probable in intradermal delivery of DNA because skin is rich in professional antigen-presenting cells, especially the dendritic cells. Condon et al. reported that through DNA immunization into skin, they were able to show expres sion of proteins encoded by DNA plasmids (18). However, such a mechanism is less likely within the muscle tissue, where there is significantly less presence of APCs. More recently, studies have reported that direct transfection of dendritic cells can occur following intramuscular inoculation DNA vaccine constructs, albeit at a lower level (19). Another study indicates that macrophages and dendritic cells may be a target cell for DNA transfected in vivo and that such a target might be important in driving immune responses in vivo (20). A clear understanding of the role of antigen-presenting cells in DNA vaccination could have important implications for this technology.

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