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
• 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
• Lipo- and polyplexes
15 min
complex
Lipo- or polyplex
mRNA
• Experimental set up
6d
Results
• Transfection efficiency of mRNA with liposomes and polymers
Transfection of Dendritic cells
with lipo- or polyplexes
Expression EGFP
and GAG
FACS
Confocal microscopy
Lipo- or polyplex
24h
Immature DC
coculture
7d
PBL
freeze
• 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).
Conclusions
Mature DC
CD14+
Blood donor (HIV+)
(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.
Phenotyping:
CD86, CD80, HLADR, CD83, CD1a,
DC-SIGN and
CCR7
Restimulation
ELISPOT with
PBL, CD4+ or
CD8+ T cells
thaw
• 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.
(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.
• Immunization of mice with lipoplexes induces Gag-specific T cell responses
• 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, Antwerp
Nationalestraat 155, B-2000 Antwerp, Belgium
Tel. +32-3-247.63.72 Fax. +32-3-216.14.31
wdehaes@itg.be
(A, B) Gag-specific IFN-g 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.