Dr. Machluf Marcelle The Faculty of Biotechnology and Food Engineering

1. Dr. Machluf Marcelle The Faculty of Biotechnology and Food Engineering Prof. Pollack Shimon The Department of Immunology Bruce Rappaport Medical School Proteo-liposomes as a new drug delivery system to HIV reservoir cells and free virion entrapment Bronshtein Tomer The Faculty of Biotechnology and Food Engineering Advisors:

2. Virus Level No inactivation of free virions Current ART’s: Why do they fail ? Anti-Retroviral Therapies Host Level Virus-Host Level • High toxicities • Low adherence • Life-time • administration • Evolution of • MDR strains. • No targeting of • reservoir cells

3. Inactivation of free virions Attachment inhibition Targeted delivery of non-antiretroviral drug to reservoir cells Syncytium inhibition Rational design of a full-scale ART ENV (gp120) CD4 co-receptor CD4+ Infected cell CD4+ Uninfected cell Delivery system must allow drug selection to fit toxicity & adherence demands

4. 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

5. 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 COS-7 co-culture BHK co-culture

6. Bio-activity of candidate model cell-lines: BHK/gp160 confocal Jurkat/CD4+/CXCR4+

7. Bio-activity of BHK model cell-line (24 hrs): BHK/gp160 Jurkat/CD4+/CXCR4+

8. 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

9. 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 • a-CCR5 monoclonal antibodies.

10. Proteo-liposomes - Protein Composition Analysis CCR5-1D4-conjugated proteo-liposomes protein composition was analyzed by immuno-blotting and FACS.

11. Proteo-liposomes - Protein Composition Analysis CCR5-1D4-conjugated proteo-liposomes protein composition was analyzed by immuno-blotting and FACS. Control: CD4-PRLP’s CD4-PRLP’s + aCCR5-RPE CCR5-PRLP’s R-PE CCR5-PRLP’s + aCCR5-RPE R-PE

12. 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).

13. 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). fully- enveloped bead Un-enveloped bead

14. Specific Fusion Between CCR5-conjugated proteo-liposomes and BHK gp160-expressing Cells Proteoliposmal cores inside BHK gp160-expressing cells Liposomes fused with BHK gp160-expressing cells. CCR5-conjugated proteol-liposomal cores localization inside BHK gp160-expressing cells.

15. Specific Fusion Between CCR5-conjugated proteo-liposomes and BHK gp160-expressing Cells

16. Control: BHK BHK + CCR5-FITC-PRLP’s Specific Fusion Between CCR5-conjugated proteo-liposomes and BHK gp160-expressing Cells BHK/gp160 FITC BHK/gp160 + CCR5-FITC-PRLP’s FITC

17. Acknowledgements Prof. Pollack Shimon The Department of Immunology Bruce Rappaport Medical School 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

18. The Bahaian gardens, Haifa - Israel