European Journal of Medical Research - Deutsche AIDS ...

June 27, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH
125
F.16 (Poster)
Addressing the human HIV-1 reservoir:
I. successful C-type lectin-specific liposomal
targeting of immature and mature myeloid
dendritic cells, macrophages, and blood monocytes
Gieseler R.K. 1 , Marquitan G. 2 , Schwarz A. 2 , Streicher H. 3 ,
Fritze A. 4 , Wader T. 2 , Hahn M. 2 , Schubert R. 4 ,
Scolaro M.J. 1
1 Rodos BioTarget GmbH, Div. of Research & Development,
Hannover, Germany, 2 LTBH Medical Research Institute, Div.
of Research & Development, Alta Loma, California, United
States of America, 3 University of Sussex, Dept. of Chemistry,
Falmer, Brighton, United Kingdom, 4 Albert-Ludwigs
University, Dept. of Pharmaceutical Technology and
Biopharmaceutics, Freiburg, Germany
Objective: Antigen-presenting cells (APCs) express C-type
lectins (CTLs) that bind infectious agents, such as HIV, by
distinctive sugars. Once internalized, HIV evades the required
process of degradation, hence establishing highly infectious
long-term viral reservoirs. HAART cannot address nonreplicative
virions. We thus aimed at designing a CTL-specific
targeting system that may deliver agents for eradicating
APC-resident HIV reservoirs.
Methods: Calcein-loaded (50 mM) DOPC/Chol/DOPE-MBP
(36.5:33.0:2.5 mol:mol:mol) 150–200 nm liposomes were
tagged with a cholesterol-linked fucose derivative (Fuc-C4-
Chol), or the respective mannose (Man-C4-Chol; pos. ctrl.) or
galactose (Gal-C4-Chol; neg. ctrl.) derivatives. Blood monocytes,
MO-derived 5-day immature or 7-day mature myeloid
dendritic cells, or 7-day macrophages generated under standard
conditions were incubated at 5x10E4–2x10E5 cells with
0.1, 1.0, or 10.0 microliters of either of the targeting systems,
for 2 or 3 h at 37°C.
Results: Fuc-4C-Chol liposomes highly specifically and efficaciously
targeted all APCs. Excellent targeting specificity,
time-dependent binding, and highly efficacious uptake of
tracer dye by all APC types was verified by fluorescence microscopy
and flow-cytometry. Besides faint cytoplasmic distribution,
calcein most strongly accumulated in the cells’ endosomal/lysosomal
system that typically harbors HIV in
reservoir cells. Intriguingly, Fuc-4C-Chol liposomes turned
out even more efficacious than Man-4C-Chol-labeled pos.
ctrls., suggesting increased CTL affinity by favorable steric
alteration of the fucosyl residue. Cellular binding of Fuc-4C-
Chol- or Man-4C-Chol-targeted liposomes was completely inhibited
by 100 mM of soluble L-fucose or D-mannose (pos.
ctrl.) [D-galactose (neg. ctrl.) was ineffective], thus clearly
demonstrating high CTL specificity. Moreover, these results
were achieved in the presence of mannose-binding lectin that,
despite being known to prevent CD209 binding of HIV, did
not hamper the targeting system’s interaction with membraneexpressed
CTLs.
Conclusions: Fuc-4C-Chol liposomes may be utilized for directional
intracellular delivery of active compounds to CTLexpressing
APCs, so as to therapeutically address chronically
infected HIV reservoir cell populations
F.17 (Poster)
Addressing the human HIV-1 reservoir:
II. Inactivation of M- and T-tropic HIV-1 strains
in myeloid dendritic cells by targeted delivery of
the HIV-agglutinating lectin Con A
Scolaro M.J. 1 , Schwarz A. 2 , Wader T. 2 , Fritze A. 3 ,
Schubert R. 3 , Gieseler R.K. 1
1 Rodos BioTarget GmbH, Div. of Research & Development,
Hannover, Germany, 2 LTBH Medical Research Institute, Div.
of Research & Development, Alta Loma, California, United
States of America, 3 Albert-Ludwigs University, Dept. of
Pharmaceutical Technology and Biopharmaceutics, Freiburg,
Germany
Objective: A fucose-dependent (Fuc-4C-Chol) C-type lectin
(CTL-)specific targeting system addressing antigen-presenting
cells (APCs) (see previous abstract), may be utilized for
intracellular endosomal/lysosomal delivery of therapeutic
compounds to HIV-1 reservoir cells. We thus inquired
whether delivery of an HIV-agglutinating lectin inactivates
such infectious HIV-1 reservoirs by employing concanavalin-
A (Con-A) that has been shown to most specifically bind alpha-Man-(1-3)-[alpha-Man-(1-6)]-Man
(mannose) cores of N-
linked oligosaccharides and to potently agglutinate HIV-1.
Methods: Fuc-C4-Chol liposomes (see previous abstract)
were loaded with horseradish peroxidase-conjugated Con-A
(0.5 mg/ml ConA/HRPO). The system was trypsinized to remove
payload potentially embedded within, or adhering to,
the liposomes’ membranes (bearing the risk to invalidate the
targeting mechanism), and the preparations were purified by
gel chromatography and kept dark at 4°C until used. Monocyte-derived
immature or mature dendritic cells were infected
with M-tropic HIV-1 Ada-M (at 67 x TCID50) or T-tropic
HIV-1 Lai (at 6.7 x TCID50) on days 2, 4, or 6. One day after
infection, thereby allowing reservoir establishment, DCs were
treated with lectin-loaded targeting system under the conditions
specified in our previous abstract.
Results: DC clustering indicating was evaluated on day 8:
while homo- and heterotypic clustering is upregulated upon
HIV-1 infection, under all conditions upon treatment with the
ConA/HRPO-loaded Fuc-C4-Chol targeting system, the clustering
behavior of DCs infected with either M- or T-tropic
HIV-1 strains was normalized. In addition, HIV-1-dependent
increased death rates of DCs were normalized upon treatment.
Moreover, the pathologic ratio between veiled-cell and dendritiform
DC morphologies upon HIV-1 infection was largely
normalized after treatment. Finally, preliminary results revealed
cessation of the deleterious transfer of HIV-1 from
DCs to T cells, complemented by PCR results verifying the
absence of T cell-integrated HIV provirus.
Conclusions: By demonstrating elimination of both M- and
T-tropic strains of HIV-1, APC-targeted delivery may pave
the way for a novel mode of treatment to eventually eliminate
chronic intracellular HIV-1 reservoirs.