Background:  Our aim was to generate DNA vectors expressing high levels of either human interleukin (IL) -15 or IL-15 receptor-alpha (IL-15Rα), and to study their in vivo biological activity as vaccine adjuvants and immune-based therapies when expressed in combination.

Methods:  Optimized DNA expression vectors for IL-15 and IL-15Rα were constructed by eliminating the instability sequences in the mRNA (RNA/codon optimization). In addition, the IL-15 signal peptide was replaced with that from tissue plasminogen activator (tPA), which dramatically increased secretion. Expression of these vectors was monitored in vitro in transfected cells, and in vivo in Balb/c mice or rhesus macaques by intramuscular injection. In mice, the DNA was alternatively delivered by hydrodynamic injection, which led to very high plasma levels (≤0.5 µg/mL). Mice were sacrificed 3 to 21 days post-injection, and muscle or plasma IL-15 measured by ELISA. The phenotype of different lymphocyte subsets in spleen, liver, lung, and gut-associated lymphoid tissue (GALT) was analyzed by multiparameter flow cytometry.

Results:  Co-expression of the optimized IL-15 and IL-15Rα greatly increased the levels of IL-15 in vivo, and resulted in approximately a 1000-fold increase compared to the wild type IL-15 cDNA alone. These data show that the complex IL-15/IL-15Rα increases the half life of the cytokine in vivo, as we have also shown in vitro. The high concentration of systemic IL-15 was biologically active, as demonstrated by the increased frequency of natural killer (NK) cells in liver and lung, and the dramatic increase of T lymphocytes with properties of effector memory cells in lung, liver and spleen. Analysis of GALT demonstrated a significant increase of CD4 T cells in lamina propria and Peyer’s patches.

Rhesus macaques injected with these vectors also express high levels of plasma IL-15, demonstrating the ability of these vectors to achieve high levels of expression in vivo.

Conclusions:  The combination of IL-15 tPA and IL-15Rα results in the mutual stabilization of the molecules, both in vitro and in vivo. The high level of expression of the optimized vectors and their enhanced activity indicate that they could be useful as adjuvants in DNA-based vaccines and in immunotherapy protocols against cancer.