Mechanisms of dna vaccineinduced immunity

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The ability to "sneak in" to an organism without inciting a strong immune response makes plasmid DNA unique among vaccines. In fact, complete adaptive immunity specific to plasmid DNA has not been reported. Importantly, although plasmid DNA is quickly deposited in myocytes and APCs (10), there is a several-hour time gap between injection of DNA vaccine and the appearance of encoded antigen. This might have major consequences for the ability of DNA vaccines to prime naive T cells. The amount of antigen produced in vivo after DNA inoculation is in the picogram to nanogram range (3). Equivalent amount of antigen introduced in vivo in the form of protein would probably be processed by macrophages without inducing an adaptive response, resulting in the impression that the antigen was either ignored or tolerized. These observations suggest that the plasmid itself may function as a powerful immunomodulator (Fig. 3).

Receptor-mediated activation of monocytes is the most likely mechanism of DNA-induced immunomodulation. Among numerous Toll-like receptors (TLRs) that function as pattern recognition receptors (PRRs) to initiate the innate immune response (114), only TLR-9 expressed on the surface of B cells, DCs, and NK cells has been shown to recognize ISSs, particularly unmethylated CpG motifs in bacterial DNA (115,116). Incorporation of a cassette containing repetitive GACGTT sequences for vaccines tested on mice, or GTCGTT in humans, may significantly boost its potency and skew the immune response toward a Th1 phenotype. The role of CpG motifs in stimulating immunity was recently reviewed (111,117,118).

Fig. 3. (A) A model DNA vaccine encoding antigen and cytokines, chemokines, and/or costimulatory molecules, promoting Th1- or Th2-type immune responses. The coexpression of specific immune molecules can influence the type of immune response against individual antigens. (B) Schematic representation of the induction of T-cell immune responses following administration of a DNA vaccine. Injection of plasmid DNA leads to transfection of professional (DC) and nonprofessional APCs (myocytes, epithelial, and endothelial cells). Following transfection, immature DCs (IDCs) become mature DCs (MDCs) and are able to prime T-cell responses. The survival and expansion of antigen-specific T cells is maintained by interactions with plasmid-transfected nonprofessional APCs.

Fig. 3. (A) A model DNA vaccine encoding antigen and cytokines, chemokines, and/or costimulatory molecules, promoting Th1- or Th2-type immune responses. The coexpression of specific immune molecules can influence the type of immune response against individual antigens. (B) Schematic representation of the induction of T-cell immune responses following administration of a DNA vaccine. Injection of plasmid DNA leads to transfection of professional (DC) and nonprofessional APCs (myocytes, epithelial, and endothelial cells). Following transfection, immature DCs (IDCs) become mature DCs (MDCs) and are able to prime T-cell responses. The survival and expansion of antigen-specific T cells is maintained by interactions with plasmid-transfected nonprofessional APCs.

Activation of signaling pathways in DCs leads to the production of not only IL-12, important for skewing immune responses toward Th1 (119), but also TNF-a and Type 1 interferons that are important for the release of IFN-y by NK cells (120). Type 1 interferons appear to be particularly important for the stimulation of Thl-type priming by ISS-containing DNA vaccines. It has been shown that IFN-a and IFN-P secreted by ISS-stimulated APCs can upregulate not only B7 but also peptide transporter associated with antigen processing (TAP) expression (121), which is essential for efficient antigen cross-presentation (14). Moreover, recently published reports indicate the importance of IL-18 and IL-23 in the Thl-polarizing capacity of DCs (122,123). Altogether, these observations might explain why gene gun delivery of plasmid DNA produces predominantly Th2-type response. Simply, gene gun forces a large portion of DNA vaccine straight into the cytoplasm, thus omitting receptors involved in DNA-induced immunostimulation. It appears that even supplying DNA vaccines with additional CpG motifs that stimulate Th1 responses cannot overrule Th2-promoting signals delivered by gene gun bombardment (124).

It becomes clear that long before plasmid-encoded protein is even expressed there is already an ongoing innate immune response able to amplify processing and presentation of the antigen by APCs. Successful immunization requires all steps of DC maturation from monocyte through IDC to MDC to occur, with each cell playing a critical role in DNA-elicited immunity (125,126). The early presence of IFN-y that is essential in stimulating maturation of monocytes and upregulation of several costimulatory and antigen-presenting molecules, such as MHC class II, B7.1/CD80, B7.2/CD86, ICAM-1/CD54, and CD40 seems to be particularly important (127). IFN-y can also significantly influence the intracellular processing of antigen by inducing replacement of standard proteasomes by immunoproteasomes that are much more efficient at producing antigenic peptides presented by MHC class I to CD8+ T cells (128,129), thus explaining the efficient priming of naïve CD8+ T cells by DNA vaccination (130). This two-phase response allowing DCs to reach a more mature phenotype characterized by the expression of several costimulatory molecules at the time of antigen presentation may also have beneficial effects through lowering the threshold of MHC/peptide-TCR binding avidity. This way more precursors including those with relatively low-affinity TCR, have a chance to be activated, thus increasing the polyclonality of T-cell responses.

The magnitude of immune induction depends upon frequency of encounters between naïve T lymphocytes and APCs within lymph nodes. Migration of directly transfected DCs to secondary lymphoid organs is critical for priming immune responses following DNA vaccination (11,15). Although the ability of naked DNA to induce migration of DCs is yet to be proved, almost certainly plasmid DNA-microparticle complexes can be phagocytosed by monocytes at the site of injection followed by migration to lymph nodes where they become perfectly functional DCs (131). DNA vaccines can be improved by including genes encoding chemokines involved in the migration of DCs. Receptors for chemokines such as CXCR4, CCR4, and especially CCR7 are upregulated in maturing DCs (132). The CCR7 ligands, secondary lymphoid chemokine (SLC/CCL21) and Epstein-Barr virus-induced molecule-1 ligand chemokine (ELC/CCL19), have been shown to be potent immunostimulators for humoral and T-cell-mediated immunity (133). Overexpression of these chemokines in plasmid-transfected cells may increase chances of direct contact between T cells and DCs accumulating along an increasing gradient of the chemokine, thus leading to improved presentation of coexpressed antigen.

Once primed, lymphocytes require favorable conditions to survive and expand. For example, Thl-type cells must reencounter antigen soon after differentiation to avoid activation-induced cell death (134). Antigen expressed in transfected muscle fibers can secure sufficient levels of antigen available to sustain Thl-type cells. Additional protection of primed T cells can be provided by properly formulated plasmid DNA. For example, 4-1BBL expressed on APCs generates signals necessary for survival of activated T cells by interaction with 4-1BB (CD137) (135). Coexpression of an antigen and 4-1BBL from the same plasmid in nonprofessional APCs may efficiently prevent deletion of antigen-specific T cells.

Persistence of low levels of antigen in the periphery may be pivotal for maintaining protective immunity. In this sense, the weakness of currently used DNA vaccines that produce small amounts of proteins might represent a beneficial role in supporting survival of memory T cells. More efficient DNA vaccines could generate substantially more antigen, but instead of improving immunization they might cause substantial detriments in the milieu of an ongoing immune response. On the other hand, low doses of presented antigen assure survival of high-avidity T cells that are crucial for an effective CTL response (136,137). This is the basis for prime-boost strategies in which DNA vaccines are followed by viral vectors.

The time lapse between entry of the transgene and expression of transgene-encoded protein results in the "unannounced" appearance of antigen in the secondary lymphoid organs. The appearance of the antigen in lymph nodes can produce sufficiently strong danger signals to initiate priming of T cells and also supports proliferation of existing memory cells leading to a strong CTL response (138,139). In fact, intralymphatic administration of DNA vaccine is 100- to 1000-fold more efficient than immunization via conventional routes (140). This observation fully supports a thesis formulated by Zinkernagel (141,142), that delivery of small amounts of antigen to secondary lymphoid organs and transient expression within the nodes is essential for induction of protective immunity. Inclusion of proper immunomodulators in DNA vaccines delivered into lymph nodes (LNs) might significantly improve this method of immunization without necessarily providing larger amounts of antigen. For example, it would be interesting to create DNA vaccines encoding an antigen and IFN-y-inducible protein-10 (IP-10), which causes retention of Th1 lymphocytes in draining LNs, thus facilitating their contact with DCs and improving presentation of weak antigens (143). This kind of vaccine formulation might be particularly useful for tumor vaccines since it combines weak TAAs with IP-10, and has already demonstrated an effective tumor-protective CD8+ T-cell response in mice (144). This is just a single example of a multigene DNA vaccine encoding an antigen and immunomodulatory molecule. To date the efficacy of at least 60 different combinations of cytokines, chemokines, and costimulatory molecules were examined in several experimental settings, with most of them being able to improve vaccine-induced immunity (72,118,145). However, evaluating the optimal composition of DNA vaccines for possible use in humans is challenging because of the enormous heterogeneity of individual host molecular and clinical profiles, which may require a more personalized approach.

Current knowledge of the mechanism by which central and effector memory cells are generated is limited (146). In fact, it has been suggested that only those vaccines that induce long-term protection exert this effect through neutralizing antibodies (147). Apparently, antigen-specific T-cell precursors induced by vaccines aimed at HIV or antigenic tumors do not persist for long enough to maintain sufficient numbers of activated effector T cells. Although the mechanisms of terminating immune responses are incompletely understood, it is possible that eradication of antigen from the host could be a major reason for termination of the immune response (142). Though this explanation might be plausible for certain kinds of transient infections, there is a little chance for complete elimination of an antigen in chronic infections or cancer. Along this line, efficient control of chronic infections might rely on a continued immune response maintained by functional memory B and T cells. Long-lasting expression of proteins provided by DNA vaccination may favor persistence of T-cell memory and consequently effective immunity (3).

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