We have used the previously developed technologies of RNA optimization to optimize expression of IL-15 cytokine, and have shown that we can over-produce bioactive cytokine after DNA delivery in mice and macaques. We explored the biology of IL-15 and showed that efficient production of IL-15 is possible only by co-expression in the same cell with the so-called IL-15 Receptor-alpha. We also showed that a second form of IL-15 (SSP IL-15) previously identified in humans and rodents as intracellular or nuclear IL-15 is also efficiently secreted from the cells when co-expressed with the IL-15 Receptor alpha. These results shed new light in the biology and regulation of IL-15 and provide methods for the efficient production and clinical application of this cytokine. Cell lines overproducing soluble bioactive IL-15/IL-15 Receptor alpha heterodimers have been constructed and were used for the production of the authentic bioactive form of IL-15 found in the body. IL-15 purified from over-producing human cells was injected in mice and shown to be bioactive. IL-15 is of interest due to its ability to stimulate the growth, activation and survival of lymphocytes, including CD8 and NK cells. Thus, IL-15 has been considered for cancer immunotherapy and for support of the growth of cytotoxic cell clones after adoptive transfer. Other proposed uses of IL-15 are in toxic shock and as vaccine adjuvant. We have shown that IL-15 injection accelerates the recovery of lymphocytes in mice rendered lymphopenic after treatment with cytotoxic drugs. We have used optimized expression vectors to express IL-12 cytokine in animals. Efficient expression results in bioactive levels, which increase immune response after DNA vaccination, thus becoming important molecular adjuvant for our vaccines. This work established methods to optimize expression of the IL-12 family of cytokines (IL-12, IL-23, IL-27, IL-35). Efficient expression of IL-27 after DNA delivery demonstrated synergy with IL-2 in the elimination of neuroblastoma metastases in mice. We have previously identified an extensive family of RNA transport elements (RTE) in the mouse genome able to replace the HIV-1 Rev/RRE posttranscriptional regulatory system, using a mutated HIV-1 DNA proviral clone as a novel molecular trap. This is general methodology for the identification of cis-acting posttranscriptional control elements in the mammalian genome. We have identified the cellular factor responsible for binding to RTE and linking it to the NXF1 export pathway. This protein, the RNA binding motif protein 15 (RBM15), had no previous assigned function. Our analysis revealed direct interaction of RBM15 and a related protein, OTT3, with the essential nuclear export factor NXF1 via their C-terminal regions. Biochemical and subcellular localization studies showed that OTT3 and RBM15 also interact with each other in vivo, further supporting a shared function. Genetic knock-down of RBM15 in mouse is embryonic lethal, indicating that OTT3 cannot compensate for the RBM15 loss, which supports the notion that these proteins, in addition to sharing similar activities, have distinct biological roles. RBM15 is an important factor facilitating the function of other proteins on RNA. The DEAD family RNA helicase Dbp5 is essential for nuclear export of mRNA and is thought to dissociate Mex67 from mRNP upon translocation, thereby generating directional passage to the cytoplasm. The molecular mechanism by which Dbp5 recognizes Mex67-containing mRNP is not clear. We found that RBM15 binds specifically to human DBP5 and facilitates its direct contact with mRNA in vivo. These results contribute significantly to the further understanding of the basic mechanisms of nucleocytoplasmic traffic of macromolecules.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Bergamaschi, Cristina; Jalah, Rashmi; Kulkarni, Viraj et al. (2009) Secretion and biological activity of short signal peptide IL-15 is chaperoned by IL-15 receptor alpha in vivo. J Immunol 183:3064-72
Petrovas, Constantinos; Chaon, Benjamin; Ambrozak, David R et al. (2009) Differential association of programmed death-1 and CD57 with ex vivo survival of CD8+ T cells in HIV infection. J Immunol 183:1120-32
Uranishi, Hiroaki; Zolotukhin, Andrei S; Lindtner, Susan et al. (2009) The RNA-binding motif protein 15B (RBM15B/OTT3) acts as cofactor of the nuclear export receptor NXF1. J Biol Chem 284:26106-16
Rosati, Margherita; Bergamaschi, Cristina; Valentin, Antonio et al. (2009) DNA vaccination in rhesus macaques induces potent immune responses and decreases acute and chronic viremia after SIVmac251 challenge. Proc Natl Acad Sci U S A 106:15831-6
Zolotukhin, Andrei S; Schneider, Ralf; Uranishi, Hiroaki et al. (2008) The RNA transport element RTE is essential for IAP LTR-retrotransposon mobility. Virology 377:88-99
Zeng, Hongkui; Horie, Kyoji; Madisen, Linda et al. (2008) An inducible and reversible mouse genetic rescue system. PLoS Genet 4:e1000069
Morrow, Matthew; Valentin, Antonio; Little, Richard et al. (2008) A splenic marginal zone-like peripheral blood CD27+B220- B cell population is preferentially depleted in HIV type 1-infected individuals. AIDS Res Hum Retroviruses 24:621-33
Bergamaschi, Cristina; Rosati, Margherita; Jalah, Rashmi et al. (2008) Intracellular interaction of interleukin-15 with its receptor alpha during production leads to mutual stabilization and increased bioactivity. J Biol Chem 283:4189-99
Rosati, Margherita; Valentin, Antonio; Jalah, Rashmi et al. (2008) Increased immune responses in rhesus macaques by DNA vaccination combined with electroporation. Vaccine 26:5223-9
Halwani, Rabih; Boyer, Jean D; Yassine-Diab, Bader et al. (2008) Therapeutic vaccination with simian immunodeficiency virus (SIV)-DNA + IL-12 or IL-15 induces distinct CD8 memory subsets in SIV-infected macaques. J Immunol 180:7969-79