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

2. 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

3. 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

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-1 89.6 ENV  Transfected cell-lines were co-cultured with Jurkat/CD4+  Viability of co-cultures was detected by Alamar-Blue TM BHK co-culture COS-7 co-culture

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

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  -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. R-PE CCR5-PRLP’s CCR5-PRLP’s +  CCR5-RPE Control: CD4-PRLP’s CD4-PRLP’s +  CCR5-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). Un-enveloped bead fully- enveloped bead

14. 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

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

16. 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

17. 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

18. The Bahaian gardens, Haifa - Israel