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