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Dr. Machluf MarcelleThe Faculty of Biotechnologyand Food Engineering
Prof. Pollack ShimonThe Department of ImmunologyBruce Rappaport Medical School
Bronshtein TomerThe Faculty of Biotechnology and Food Engineering
Proteo-liposomes as a new drug delivery system to HIV reservoir cells and free
virion entrapment
Advisors:
Current ART’s: Why do they fail ?
Host Level
• High toxicities
• Low adherence
• Life-time
administration
Virus Level
No inactivation
of free virions
Virus-Host Level
• Evolution of
MDR strains.
• No targeting of
reservoir cells
Anti-Retroviral Therapies
Rational design of a full-scale ART
CD4+Infected
cell
CD4+Uninfected
cell
ENV (gp120)
co-receptor
Inactivation of free virions
Attachment inhibition
Syncytium inhibition
Targeted delivery of non-
antiretroviral drug to reservoir cells
Delivery system must allow drug selection to fit toxicity & adherence demands
CD4
Long term goal
To develop CCR5 conjugated proteo-liposomal
constructs for targeted drug delivery &
inactivation of free virions
Loaded with a chemotherapeutic drug
I.V. administration
sCD4
Bio-activity of candidate model cell-lines:
BHK & COS-7 cell-lines were transfected with HIV-189.6 ENV
Transfected cell-lines were co-cultured with Jurkat/CD4+
Viability of co-cultures was detected by Alamar-BlueTM
0
20
40
60
80
100
0 3 25
co-culture incubation time [hr]
% V
iabi
lity
of c
ontr
ol
0
20
40
60
80
100
0 3 25
co-culture incubation time [hr]
% V
iabi
lity
of c
ontr
ol
BHK co-culture COS-7 co-culture
Bio-activity of candidate model cell-lines:
BHK/gp160
Jurkat/CD4+/CXCR4+
confocal
Bio-activity of BHK model cell-line (24 hrs):
BHK/gp160
Jurkat/CD4+/CXCR4+
Bio-activity of BHK model cell-line (24 hrs):
BHK control cells and BHK/gp160 model cells were incubated
with sCD4-FITC-conjugated beads and analyzed by FACS
BHK + CD4-FITC
BHK/gp160 + CD4-FITC
FITC
Analysis of CCR5 Expression by Cf2Th Cells
• Cf2th/CCR5 cells over expressing human CCR5
were cultured and harvested.
• Expression level of CCR5 was analyzed with 1D4
-CCR5 monoclonal antibodies.
Proteo-liposomes - Protein Composition Analysis
CCR5-1D4-conjugated proteo-liposomes protein
composition was analyzed by immuno-blotting and FACS.
Proteo-liposomes - Protein Composition Analysis
CCR5-1D4-conjugated proteo-liposomes protein
composition was analyzed by immuno-blotting and FACS.
R-PE
CCR5-PRLP’s
CCR5-PRLP’s + CCR5-RPE
Control: CD4-PRLP’s CD4-PRLP’s + CCR5-RPE
R-PE
Proteo-liposomes morphology
CCR5-1D4-conjugated proteo-liposomes were analyzed
by fluoresce microscopy. Beads are labeled with BSA-
FITC (green), lipid membrane labeled with DiI (red).
Proteo-liposomes morphology
CCR5-1D4-conjugated proteo-liposomes were analyzed
by confocal scanning microscopy. Beads are labeled with
BSA-FITC (green), lipid membrane labeled with DiI (red).
Un-enveloped bead fully-enveloped bead
Specific Fusion Between CCR5-conjugated
proteo-liposomes and BHK gp160-expressing Cells
Liposomes fused with BHK gp160-expressing cells.
CCR5-conjugated proteol-liposomal cores localization inside BHK gp160-expressing cells.
Proteoliposmal cores inside BHK gp160-expressing cells
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
1.E-04 1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06
CD4 molecules per cell
% c
ells
fu
sed
wit
h l
ipo
so
mes
BHK/gp160 (model) BHK (control)
Specific Fusion Between CCR5-conjugated
proteo-liposomes and BHK gp160-expressing Cells
BHK/gp160
BHK/gp160 +CCR5-FITC-PRLP’s
FITC
Specific Fusion Between CCR5-conjugated
proteo-liposomes and BHK gp160-expressing Cells
Control: BHK BHK + CCR5-FITC-PRLP’s
FITC
Acknowledgements
Dr. Machluf Marcelle
The Faculty of Biotechnology
and Food Engineering
Dr. Efrat Goren
Dr. Vered Kivity
Ofra Benny
Limor Baruch
Maayan Duvshani Eshet
Yuval Eitan
Koby Gvilli
Amit Goren
Nizan Dahan
Prof. Pollack Shimon
The Department of Immunology
Bruce Rappaport Medical School
The Bahaian gardens, Haifa - Israel