INTRODUCTION

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In 1990, it was first reported that intramuscular injection of plasmid DNA in a simple saline solution could transfect muscle cells in vivo (1). This observation suggested that plasmid DNA could be used to express foreign proteins inside a cell, and thus also potentially to induce an immune response against the expressed proteins. It is now well established that this technology can induce antibodies, helper T cell responses, cytotoxic T cell (CTL) responses, and protective immunity in various animal models (for a review see Donnelly and coworkers [2]). Several features of DNA vaccines have made them an attractive alternative to conventional methods of vaccination. These include the ability of DNA vaccines to: (1) express native protein antigens in situ for recognition by B cells and presentation by MHC class I and II molecules to prime helper T cells and CTLs, (2) elicit robust immune responses in many animal species, and (3) be efficiently manufactured and well characterized. Much is still being learned about the mode of action of DNA vaccines in animals and humans. However, a reasonable hypothesis involves a combination of cross-priming, via transfer of antigen from transfected muscle cells, and direct transfection of antigen-presenting cells (APCs). Elucidation of the precise mechanisms of immune priming will be important in the development of effective DNA vaccines, as rational approaches to designing improved DNA vaccines may be required.

	MECHANISMS OF CTL INDUCTION

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One key to improving DNA vaccination is to better understand the nature of the APCs responsible for the induction of immune responses, particularly in CTLs. Although the cellular mechanisms by which antigens are processed for MHC-restricted presentation to T lymphocytes are well known, the cooperative interactions between somatic cells and antigen-presenting cells for in vivo antigen presentation are still being elucidated.

DNA vaccines induce potent CD8-restricted CTL responses in mice against antigens from viruses, bacteria, parasites, and tumors. This is true for antigens targeted to the cytoplasm, nucleus, or endoplasmic reticulum for secretion or membrane insertion. The structures of DNA vaccine-encoded antigens capable of inducing a CTL response include whole protein, truncated protein, fusion with another protein, a string of several CTL peptides, a CTL peptide embedded in a heterologous protein, and a minimal CTL peptide. The induction of CTLs by DNA vaccines in mice requires CD28 costimulation (3) and CD4⁺ helper T cell responses (4), suggesting that established mechanisms of T cell priming are involved. That DNA vaccines encoding minimal epitopes can induce CTLs, however, suggests that CD4⁺ helper T cell-independent CD8⁺ T cell responses may be mediated by direct or indirect activation of APCs by the bacterially derived plasmid or CpG motifs derived therefrom (5).

Secreted proteins can be endocytosed by several types of APCs and ultimately presented via MHC class II molecules. Antigens, such as viral proteins, that are presented via MHC class I molecules to naive CD8⁺ T lymphocytes appear to use more complex pathways. Some viruses may infect "professional" APCs directly. But, a growing body of evidence supports the ability of immature dendritic cells (DCs) to endocytose soluble proteins and debris from apoptotic cells, and then present these antigens in the context of MHC class I molecules after differentiating to mature DCs (6).

In the case of intramuscularly injected DNA vaccines, myocytes appear to be the predominant cell type transfected, and yet this method of immunization yields potent cell-mediated protective immunity. Therefore, antigen transfer from myocytes to "professional" APCs may be of importance in the ability of intramuscularly administered DNA vaccines to induce CTL responses. The production of antigen by muscle cells alone is sufficient to induce CTL responses. We found that when C2C12 myoblasts that had been stably transfected with the gene for influenza nucleoprotein (NP) were transplanted into histocompatible C3H mice, an H-2K^k-restricted CTL response was induced, even though synthesis of the antigen was limited to the transplanted myoblasts (9). Similar observations were made after implantation of transduced tumor cells (10).

Several studies have shown that the principal antigen-presenting cells responsible for induction of CTLs after intramuscular injection of DNA vaccines appear to be derived from the bone marrow. The ability of antigens produced by non-APCs to prime CTLs in the context of MHC molecules present only on "professional" APCs, termed cross-priming, has been demonstrated in parental  F₁ mouse bone marrow chimeras (10). When CT26 colon adenocarcinoma cells (H-2^d) that had been transfected with the gene for influenza NP were used to immunize B6  CB6F1 bone marrow chimeras, an H-2D^b-restricted CTL response was induced although the CT26 cells did not express H-2D^b. Similarly, when we transplanted C2C12 myoblasts (H-2K^k) into (BALB/c × C3H) F₁ mice, CTL responses were obtained that recognized distinct peptide epitopes presented by both H-2K^k (NP 50-57) and H-2K^d (NP 147-155), even though the myoblasts did not express H-2K^d (9). These responses were observed whether the myoblasts were transplanted intramuscularly, or intraperitoneally, where they would not be expected to fuse with host cells. Both of these studies support the hypothesis that antigens (or their epitopes) synthesized in non-APCs can be transferred to APCs for presentation in the context of MHC class I. When parental  F₁ bone marrow chimeras were injected intramuscularly with plasmid DNA, CTL responses were seen only to the peptide presented by the MHC class I molecules found on the donor bone marrow (11). Similar results were found when SCID mice were reconstituted with semiallogeneic bone marrow (14). In these studies, when reconstitution with semiallogeneic APCs was performed weeks after the injection of DNA, a CTL response restricted by MHC molecules of the transplanted APCs still was observed. This indicates that direct transfection of APCs by the plasmid is not required because it is unlikely that free plasmid could have existed at that time. Thus, not surprisingly, for DNA vaccines, MHC molecules on APCs play a dominant role in the induction of CTL responses, while somatic cells appear to be inefficient at presenting antigen in the context of MHC class I in the absence of alloantigenically matched APCs.

The cellular mechanisms by which cross-priming occurs are still undefined, but involve the internalization and intracellular processing of exogenous protein antigens by APCs for presentation by MHC class I molecules and priming of CTL responses (15). The source of antigen may be inoculated protein (such as in a vaccine), proteins from a circulating pathogen, or proteins released by cells (such as during an infection). This process may involve whole protein, fragments thereof, complexes with heat shock proteins (16), or apoptotic bodies from dying cells (7). A specialized subset of APCs has the ability to internalize and process these antigens in a manner distinct from the classic pathway of MHC class I presentation of newly synthesized proteins. Delivery of epitope peptides to MHC class I molecules in cross-priming requires that the bone marrow-derived APCs express functional transporter-associated peptide (TAP)-encoding genes. Bone marrow chimeras made by grafting TAP^-/- bone marrow into TAP^+/+ recipients are unable to respond to NP expressed in transfected CT26 cells (17). In contrast, these chimeras were able to respond to recombinant vaccinia virus expressing an NP peptide minigene linked to a signal peptide that provides for translocation to the endoplasmic reticulum (ER). Thus, in cross-priming, unprocessed protein antigens may be delivered to APCs for processing. Intramuscular vaccination with influenza NP DNA induced CTLs specific for the known dominant epitope peptides NP50-57 in H-2^k mice, NP147-155 in H-2^d mice, and NP366- 372 in H-2^b mice (9, 18), as well as subdominant naturally processed peptides. A plasmid encoding a mutant form of NP in which the two anchor residues of the 147-155 epitope had been mutated induced CTL responses against a subdominant epitope, 218-226, in BALB/c mice. CTLs that recognized this epitope were able to kill target cells infected with influenza virus, indicating that the peptide or a closely related epitope is naturally processed and presented on MHC class I molecules in influenza virus infection (18).

Loirat and coworkers (19) have shown that a DNA vaccine vector driven by a muscle-specific promoter was capable of inducing a full range of immune responses, including CTLs, in mice. Hence, because CTLs were primed by a DNA vaccine that expressed antigen only in a non-APC (i.e., muscle cells), regardless of which cells internalized the plasmid, indicates that cross-priming was involved. Corr and colleagues (20) have demonstrated that, at least for syringe injection of DNA, both direct transfection of APCs and non-APCs, followed by cross-priming, are involved in CTL priming. However, using the techniques of regulated gene expression and bone marrow chimeras, they concluded that cross-priming played the major role in the magnitude of CTL responses. Taken together, these lines of evidence indicate that cross-priming of CTL responses can occur by expressing antigens in non-APCs (such as muscle cells) and that expression of antigens in APCs is not required for induction of CTLs by DNA vaccines.

Unlike vaccination by intramuscular injection of DNA, vaccination by particle bombardment transfects cells of the epidermis and dermis by direct deposition of DNA-coated gold beads. Therefore, Langerhans' cells, the resident "professional APCs" of the skin, may be directly transfected. Indeed, Condon and coworkers (21) have shown that APCs are transfected by gene gun administration of DNA vaccines, with subsequent migration of the cells to the draining lymph node. It has been proposed that this mechanism is the predominant means of priming CTLs by the gene gun (22). Migration of Langerhans' cells to the regional lymph node is thought to initiate the immune response to the expressed protein. In this model, both TAP-dependent and TAP-independent processing may occur. A construct encoding a minimal peptide epitope induced CTLs when delivered by gene gun if the peptide was linked to an ER translocation signal sequence (23). A similar phenomenon has been observed for some peptides delivered as vaccinia virus recombinants (17). Full-length proteins encoded by plasmids administered by gene gun appear to induce CTLs that recognize naturally processed peptides, and thus likely have been processed in a TAP-dependent manner (23).

	TARGETING OF DNA TO APCs

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Presentation of antigens by dendritic cells (DCs) is a potent stimulus to immune responses, particularly to cell-mediated immunity and the development of CTLs. DCs pulsed with peptides derived from tumor antigens or transfected ex vivo with genes encoding tumor antigens elicit highly potent antitumor immune responses in laboratory animals (6), and this approach is now being attempted in clinical studies of cancer immunotherapy. Studies of DNA vaccines have shown that bone marrow-derived antigen-presenting cells play a pivotal role in the induction of cytotoxic T cell responses by both intramuscular and gene gun delivery (9, 11, 21, 24). The relative contributions of transfer of antigen from transfected muscle cells, and of direct transfection of APCs, to the presentation of antigen after intramuscular injection of plasmid DNA, are undetermined at present.

DCs transfected with DNA vaccines in vitro are potent inducers of immune responses in vivo (24). There is evidence (direct and indirect) in support of transfection of APCs after DNA vaccination by syringe injection. Indirect evidence is as follows. First, as with skin, muscle tissue can be removed shortly after intramuscular injection of DNA vaccines without detriment to the development of immune responses, including CTLs (27). Therefore, transfection of cells distal to the injection site (possibly APCs) is sufficient to prime CTLs. Second, DCs isolated from DNA-vaccinated muscle have been shown to present antigen in vitro to T cell hybridomas (28), indicating that these cells either acquired protein antigen or were transfected by the DNA vaccine. Third, DNA vaccines designed to express rapidly degraded ubiquitin-antigen fusion proteins were able to prime CTLs but not antibodies (29). Therefore, these DNA vaccines provided few, if any, of the extracellular proteins available for cross-priming. A reasonable explanation of these data is that APCs were transfected, with CTL epitopes from the rapidly degraded proteins being presented by newly synthesized MHC class I molecules. However, it cannot be ruled out that CTLs were primed by expression of antigen in non-APCs followed by transfer of the peptide epitopes to APCs, possibly in a complex with heat shock proteins. More direct evidence of transfection of APCs has been demonstrated by the presence of antigens or reporter genes in such cells in situ after DNA vaccination. Chattergoon and coworkers (30) and Akbari and colleagues (31) detected green fluorescent protein in macrophages and complement C5 in DCs, respectively, after vaccination with DNA encoding these proteins. Bouloc and coworkers (26) provided the most definitive evidence of transfection of APCs by demonstrating mRNA encoding the DNA vaccine antigen in these DCs. Furthermore, these DCs isolated from injected skin tissue were able to stimulate proliferation of antigen-pulsed T cells in vitro and to prime CTLs in vivo after adoptive transfer to naive mice. Taken together, these data support the hypothesis that DNA vaccines can transfect APCs, leading to priming of CTL responses. This is true for both the gene gun and syringe injection modes of administration. However, the efficiency of transfection, at least with syringe injection, is probably low, because of the requirement for cellular uptake of DNA. Therefore, methods to enhance transfection of APCs will likely lead to more potent DNA vaccines and targeting of APCs for transfection by DNA vaccines is a logical approach to increasing DNA vaccine potency.

The potency of antigens presented directly by DCs and Langerhans cells in inducing CTL responses has made targeting of these cell types a research priority for DNA vaccination against both infectious diseases and cancer. The extent to which DCs may be transfected directly by intramuscular injection of plasmid DNA may depend on the excipients used in the particular formulation of the plasmid. Various ligands specific for receptors on DCs are potential methods for targeting DNA vaccines directly to DCs (32). In addition, it may be possible to increase delivery of DNA to APCs in vivo nonspecifically using a particulate formulation of DNA, in order to take advantage of the high phagocytic capacity of such cells, particularly immature dendritic cells. In support of this hypothesis, intramuscular injection of poly(lactide-co-glycolide) (PLG) microspheres ~1 µm in diameter with surface-adsorbed DNA induced substantially higher levels of immune responses compared with naked DNA (33). The mode of action of these particles has not yet been fully elucidated. However, preliminary results indicate that these particles do not substantially increase expression in muscle in vivo (33) but can transfect dendritic cells in vitro, resulting in processing and presentation of antigen to T cells (Denis-Mize, K. S., M. Dupuis, M. L. Mackichan, M. Singh, D. O'Morgan, J. B. Ulmer, J. Donnelly, D. MacDonald, and G. Ott, manuscript submitted for publication). Therefore, targeting of DNA vaccines to both APCs and non-APCs may have beneficial effects, although probably through different mechanisms, such as expression of antigen within APCs versus cross-priming, respectively.

Viral vectors, such as the alphaviruses Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus, can be generated that encode an antigen of interest, but do not produce viral structural proteins or infectious particles in vivo. These vectors use the novel viral RNA-dependent RNA polymerase to amplify multiple copies of mRNA encoding the gene of interest within the cytoplasm of an infected cell (for review see Schlesinger and Dubensky [34]). Furthermore, certain alphaviruses have tropism for DCs, making this approach particularly attractive for induction of cell-mediated immunity. Hence, alphavirus vectors encoding viral and tumor antigens have been shown to induce potent immune responses in animal models (35). In addition, DNA-based vaccines encoding the RNA replicon of alphaviruses can also be used to elicit immune responses. These consist of conventional plasmid DNA vectors with a gene cassette containing the nonstructural proteins and a gene of interest. In this way, the transcript expressed off the conventional plasmid drives a self-amplifying RNA replicon. Data generated so far indicate that these DNA vaccines may be more potent than conventional DNA vaccines (38, 39). Reasons for this may include: (1) higher levels of antigen expression; (2) the presence of a double-stranded RNA intermediate, which can have immunostimulatory effects; (3) additional helper T cell epitopes present in the nonstructural proteins; and (4) facilitation of cross-priming through induction of apoptosis of cells expressing the nonstructural proteins. Therefore, the use of replication-incompetent virus particles (replicons) or vectors to deliver either DNA or RNA vaccines has potential for facilitating immune priming in two ways: cross-priming and direct targeting of DCs.