Dendritic cell delivery of plasmid DNA. Applications for controlled genetic immunization.

Positive human clinical data using biolistic-mediated gene transfer (i.e., gene gun) to administer a nucleic acid-based Hepatitis B vaccine has validated genetic immunization as an effective clinical vaccine modality. Although the precise mechanism of action has yet to be determined, preclinical studies using jet injection have indicated that direct targeting of resident antigen presenting cells (Langerhan's cells) in the skin as the primary immunological driving force for the potent and long-lived immune response. Moreover, positive results with topical delivery of genetic vaccines and ex vivo loading of dendritic cells with antigen has strengthened the movement toward directly targeting antigen presenting cells as a means to amplify, control, and mediate the immunological consequences of prophylactic and/or therapeutic genetic vaccines. Despite these encouraging results with the gene gun, it is unclear whether this technology will translate into commercially available vaccines due to potential product development barriers such as cost and convenience. It is clear that safety concerns in using genetic approaches to treat and prevent disease have highlighted the need for strict product requirements for genetic vaccines. A plausible strategy to meet these requirements is to combine controlled plasmid delivery systems with tissue-specific gene expression systems.

Controlled plasmid delivery and gene expression : applications for nucleic Acid-based vaccines.

After the concept of genetic immunization was first demonstrated by Johnston's group in 1992 (1), numerous studies have reported the potential prophylactic and therapeutic use of nucleic acid-based vaccines for combating various infectious diseases (2-4). Vaccines of this composition appear to be both efficacious in the short term, and able to elicit a prolonged anamnestic response capable of preventing or resolving infection when challenged at up to one year after vaccination (5). Nucleic acid-based vaccines elicit a broader immune response than do subunit vaccines, inducing both cellular and humoral responses that are reminiscent of attenuated and whole-killed viral vaccines. Further, nucleic acid-based vaccines can be prepared with relative ease of synthesis and production. Expression plasmids can be generated quickly once the antigen's coding sequence is known and small- and large-scale purification methods are well established. Nucleic acid-based vaccines also avoid some of the safety concerns of conventional vaccines in that there is no chance of disease due to co-purification of contaminating virus or reversion of the attenuated strain in the patient. This is not to claim that the safety issues surrounding nucleic acid-based vaccines are minimal. The major theoretical concerns surrounding the safety of this technology include plasmid integration into the host genome, transformation of somatic or stem cells, and tolerability. However, there is no published evidence that administration of unformulated or 'naked' plasmid produces a severe short or long term deleterious effect (6).

Lymphoid signal transduction mechanisms linked to cellular prion protein.

The normal cellular isoform of the prion protein (PrPC) is a glycosylphosphatidylinositol-anchored cell surface protein that is expressed widely, including in lymphoid cells. We compared lectin-induced mitogenesis and selected cell signaling pathways in splenocytes from wild-type BALB/c mice and Zrch Prnp0/0 (PrP0/0) mice bred on a BALB/c background for more than 10 generations. 3H-thymidine incorporation induced by concanavalin A (Con A) or phytohemagglutinin (PHA) was significantly reduced in PrP0/0 splenocytes, most prominently early in activation (24 and 48 h). Con A activation in PrP0/0 splenocytes was associated with differences in the phosphorylation (P) patterns of protein kinase C (PKC alpha/beta, but not delta) and the PKC downstream effectors p44/42MAPK (mitogen-activated protein kinase). P-PKC and P-MAPK profiles were similar in wild-type and PrP0/0 splenocytes following PMA treatment, indicating that the ability of these 2 enzymes to be phosphorylated is not impaired in the absence of PrPC. Con A-induced calcium fluxes, monitored by indo-1 fluorescence, were equivalent in PrP0/0 and PrP+/+ splenocytes, suggesting that calcium-dependent mechanisms are not directly implicated in the differential phosphorylation patterns or mitotic responses. Our data indicate that PrP0/0 splenocytes display defects in upstream or downstream mechanism(s) that modulate PKCalpha/beta phosphorylation, which in turn affects its capacity to regulate splenocyte mitosis, consistent with a role for PrPC in immune function.

Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells. Essential roles of a variant NF-kappa B site and p65 homodimers.

Intercellular adhesion molecule-1 (ICAM-1) is greatly up-regulated on endothelial cells at sites of inflammation and is involved in leukocyte attachment and extravasation. Previously, we had shown that the ICAM-1 gene expression in human umbilical vein endothelial cells (HUVECs) was transcriptionally regulated by tumor necrosis factor-alpha (TNF-alpha) (Wertheimer, S. J., Myers, C. L., Wallace, R. W., and Parks, T. P. (1992) J. Biol. Chem. 267, 12030-12035). In the present investigation, TNF-alpha-induced transcription was found to be initiated exclusively at two sites, 40 and 41 base pairs upstream of the translation start site. Deletion analysis of the 5' regulatory region of the ICAM-1 gene revealed a 92-base pair sequence which was both necessary and sufficient to confer TNF-alpha responsiveness to a linked luciferase reporter gene in transient transfection assays. This TNF-alpha-responsive region contained a variant NF-kappa B site at -187 to -178, which when mutated, completely abolished ICAM-1 promoter activation by TNF-alpha, interleukin-1 beta, and lipopolysaccharide. Two inducible nuclear protein complexes bound to the ICAM-1 kappa B and were identified as the NF-kappa B p65 homodimer and p65/p50 heterodimer. Overexpression of p65, but not p50, transactivated the ICAM-1 promoter in a kappa B site-dependent manner in HUVECs. In addition, p65-mediated transactivation was suppressed by co-expression of p50. Our results suggest that cytokine activation of the ICAM-1 promoter in HUVECs may critically depend on p65 homodimers binding to a variant kappa B site.

Synergistic effect of formulated plasmid and needle-free injection for genetic vaccines.

PURPOSE: A plasmid-based gene expression system was complexed with protective, interactive, and non-condensing (PINC) polymer system and administered with Medi-Jector, a needle-free injection device (NFID), to achieve high and sustained levels of antigen-specific antibodies in blood circulation. METHODS: Human growth hormone (hGH) or bacterial beta-galactosidase gene expression plasmids driven by a cytomegalovirus (CMV) promoter were formulated in saline or complexed with a PINC polymer, polyvinylpyrrolidone (PVP), and intramuscularly or subcutaneously administered into dogs and pigs using a 22-gauge needle or a NFID. The hGH-specific IgG titers in serum were measured by an ELISA. Beta-galactosidase expression was measured in injected muscles by an enzymatic assay or immunohistochemistry. The effect of NFID on DNA stability and topology was assessed by gel electrophoresis. RESULTS: Intramuscular (i.m.) or subcutaneous (s.c.) injection of a hGH expression plasmid pCMV-hGH (0.05-0.5 mg/kg) in dogs and pigs elicited antigen-specific IgG antibody titers to expressed hGH. With both routes of injection, pDNA delivery by a NFID was superior to pDNA injection by needle. The magnitude of hGH-specific IgG titers with NFID was 15-20-fold higher than needle injection when pDNA was complexed with PVP, and only 3-4-fold higher with pDNA in saline. The transfection efficiency in the injected muscle, as measured by beta-galactosidase expression, following i.m. injection of pCMV-betagalactosidase/PVP, was not significantly different between needle and NFID-injected groups. CONCLUSIONS: These data demonstrate that the combination of pDNA/ PVP complexes and a NFID act synergistically to achieve high and sustained levels of antigen-specific IgG response to expressed antigen. This gene delivery approach may offer advantage over needle injection of naked DNA for the development of genetic vaccines.