Materials and methods

1. Whistler, Canada, 20-25.03.2011, Keystone “HIV evolution, genomics and pathogenesis” Liposomal Transfection of Dendritic Cells with gag mRNA Stimulates HIV-1 Specific Immune Responses Winni De Haes1, Charlotte Pollard1,4,Céline Merlin1, Joanna Rejman3, Stefaan De Smedt3, Johan Grooten4, Guido Vanham1,2 Stefaan De Koker1,4& Ellen Van Gulck1 1Virology unit, Department of Microbiology, Institute of Tropical Medicine Antwerp (ITMA), Belgium, 2Department of Biomedical Sciences, Faculty of Pharmaceutical, Veterinary and Biomedical Sciences, University of Antwerp, 3Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Belgium, 4Laboratory of Molecular Immunology, Departement of Molecular Biomedical Research, Ghent University, Belgium Background Materials and methods • Lipo- and polyplexes • 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 15 min Transfection of Dendritic cells with lipo- or polyplexes Lipo- or polyplex complex mRNA • Experimental set up Expression EGFP and GAGFACS Confocal microscopy 6d Lipo- or polyplex Phenotyping: CD86, CD80, HLA-DR, CD83, CD1a, DC-SIGN and CCR7 Results Mature DC 24h Immature DC • Transfection efficiency of mRNA with liposomes and polymers CD14+ Blood donor (HIV+) RestimulationELISPOT with PBL, CD4+ or CD8+ T cells coculture 7d PBL freeze thaw (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. • Functional capacity of DC transfected with lipofectamine and gag mRNA to stimulate GAG specific T cell responses (A, B) DCs transfected with Lipofectamine and mRNA encoding for GAG (LFegfp) or DCs electroporated with gag mRNA (EPgag) similarly expand IFN-g and IL-2 secreting autologous PBL after a seven day coculture. • 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-a, PGE2, IL-6, IL-1b). (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-g and IL-2 secreting CD4+ and CD8+ T cells. Conclusions • Immunization of mice with lipoplexes induces Gag-specific T cell responses (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. • 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-g 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 Institute of Tropical Medicine, AntwerpNationalestraat 155, B-2000 Antwerp, BelgiumTel. +32-3-247.63.72 Fax. +32-3-216.14.31wdehaes@itg.be