Liposomal Transfection of Dendritic Cells with gag mRNA Stimulates HIV-1 Specific Immune Responses Winni De Haes 1, Charlotte Pollard 1,4,Céline Merlin 1, Joanna Rejman 3, Stefaan De Smedt 3, Johan Grooten 4, Guido Vanham 1,2 Stefaan De Koker 1,4 & Ellen Van Gulck 1 1 Virology unit, Department of Microbiology, Institute of Tropical Medicine Antwerp (ITMA), Belgium, 2 Department of Biomedical Sciences, Faculty of Pharmaceutical, Veterinary and Biomedical Sciences, University of Antwerp, 3 Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Belgium, 4 Laboratory of Molecular Immunology, Departement of Molecular Biomedical Research, Ghent University, Belgium Institute of Tropical Medicine, Antwerp Nationalestraat 155, B-2000 Antwerp, Belgium Tel Fax Infection with human immunodeficiency virus type 1 (HIV-1) is characterized by dysfunction of HIV-1-specific T cells Dendritic cells (DCs) are potent antigen-presenting cells that hold promise to boost and broaden HIV-specific T cell responses gag mRNA encodes for structural and matrix proteins that constitute the virion capsid and envelope from HIV-1. It is immunodominant and relatively conserved DCs electroporated with gag mRNA from HIV can expand and broaden HIV-specific T-cell responses in vitro (Van Gulck et al. Blood 2006, JVI 2008) The ex vivo loading of DCs is labour-intensive and time-consuming Liposomes (Lipofectamine, DOTAP/DOPE) and polymers (Linear polyethyleneimine, JetPEI and in vivo-jetPEI) are potential delivery vehicles for nucleic acids Dendritic cells incubated with gag mRNA alone failed to upregulate the expression of maturation markers. Lipofectamine alone slightly increased expression of the lymph node migration marker CCR7, the maturation marker CD83 and the co-stimulatory molecules CD80 and CD86. mRNA/Lipofectamine lipoplexes more pronouncedly increased the expression of the membrane markers.Transfected DCs could still be further matured by adding the standard Jonuleit cocktail (TNF- , PGE 2, IL-6, IL-1  ). (A, B) DCs transfected with Lipofectamine and mRNA encoding for GAG (LFegfp) or DCs electroporated with gag mRNA (EPgag) similarly expand IFN-  and IL-2 secreting autologous PBL after a seven day coculture. Materials and methods Background Conclusions Twenty-four hours after transfection, 32% DCs express eGFP or GAG protein after transfection with lipoplexes containing Lipofectamine and mRNA. DCs transfected with Lipofectamine complexed with gag mRNA showed small upregulation of maturation membrane markers: CD80, CD83 and CD86. These DCs can expand the IFN-  and IL-2 responses of both CD4+ and CD8+ T cells after seven days of coculture. Mice immunized with lipoplexes induced Gag specific T cell responses from cells isolated from spleen ad lymph nodes Functional capacity of DC transfected with lipofectamine and gag mRNA to stimulate GAG specific T cell responses Results Transfection efficiency of mRNA with liposomes and polymers (A) Expression efficiencies evaluated by flow cytometry. Liposomes (Lipofectamine, LF and DOTAP/DOPE) and poymer linear PEI (jetPEI and in vivo-jetPEI) were used to complex mRNA encoding either EGFP (green bars) or GAG (red bar). (B) Kinetics of the protein expression after transfection with lipoplexes containing Lipofectamine and mRNA encoding for GAG protein. (C) Confocal image of DCs transfected with lipofectamine and mRNA encoding for EGFP. The nucleus of the DCs is stained in blue with DAPI. Immunization of mice with lipoplexes induces Gag-specific T cell responses Lipo- and polyplexes Experimental set up Blood donor (HIV + ) freezethaw coculture 7d 24h 6d Phenotyping: CD86, CD80, HLA- DR, CD83, CD1a, DC-SIGN and CCR7 PBL CD14+ Immature DC Mature DC complex mRNA Lipo- or polyplex 15 min Transfection of Dendritic cells with lipo- or polyplexes Lipo- or polyplex Expression EGFP and GAG FACS Confocal microscopy Restimulation ELISPOT with PBL, CD4+ or CD8+ T cells (C-F) After coculture CD4+ end CD8+ T cells were isolated with MACS miltenyi beads and assayed in ELISPOT. Lipofectamine transfected DCs were clearly capable of expanding IFN-  and IL-2 secreting CD4+ and CD8+ T cells. (A, B) Gag-specific IFN-  secreting cells isolated from speen and lymph nodes were observed for mice immunized with lipoplexes as compared to immunization with naked gag mRNA. This demonstrates that lipoplexes can be successfully used to deliver mRNA and prime immune responses in vivo. Whistler, Canada, , Keystone “HIV evolution, genomics and pathogenesis”