1. Candidate Microbicides: What we can learn from in vitro work Guido Vanham, MD PhD gvanham@itg.be Institute of Tropical Medicine, Antwerp

2. Vaginal HIV transmission

3. TRANSCRIPTION + TRANSLATION REVERSE TRANSCRIPTASE INHIBITORS BINDING INHIBITORS FUSION INHIBITORS INTEGRASE INHIBITORS ssRNA dsDNA HIV TARGET CELL CD-4 CCR-5 ITM – Y. Van Herrewege Direct DISRUPTION HIV life Cycle Potential targets for prevention

4. Possible classes of candidate microbicides - Buffers: Acidform, Buffergel: still in trial but only indirect antiviral action –Virus disrupters: Nonoxynol-9, Savvy (C31G) = obsolete –Non-specific binding inhibitors: Cellulose sulphate; Carraguard, PRO-2000; Vivagel some failed, some still in trial but even in vitro weak anti-HIV activity –Inhibitors of gp120:CD4 (e.g. BMS806, BMS793) –Inhibitors gp120:CCR5 (e.g. TAK-779, Maraviroc) –Inhibitors of gp120: DC SIGN (e.g. Mannan) –Fusion inhibitors (e.g. T20, D-peptides) –Reverse Transcriptase inhibitors (RTI): in trial Nucleotide RTI: PMPA (Tenofovir) + FTC (Truvada) NNRTI: TMC120 (Dapivirin), UC781 –Integrase inhibitors e.g. L 870 812 (Raltegravir analogue) ? –Protease inhibitors e.g. Saquinavir ?

5. In Vitro Activity against pathogen Cellular toxicity profile Animal models Safety: - Rabbit vaginal irritation Efficacy to prevent infection: - NOD/SCID-PBL mice: HIV - Macaques: (SIV or SHIV) Human (clinical) Safety - In low-risk women (Phase I) - In representative population (Phase I/II) Effectiveness (Phase III) 10 + years Clinical Research Process

6. In Vitro models to test HIV Microbicides - Limited access (HT -) - Risk of damaging epithelium Main advantagesMain disadvantages CD4/CCR5 (+) cell lines e.g. GHOST, U87, TZMbl - Rapid screening - Single cycle virus (no L3 required) Cells not representative for in vivo targets Mitogen activated PBMC- Standard system - Relatively quick + easy Only activated T cells Co-cultures of dendritic cells and T cells (DC/T4) More representative for primary targets in sexual mucosa More complex and time consuming manipulation DC/T4 + epithelial cellsAdditional relevanceAdditional complexity Cervico-vaginal explantMost representative- Limited access - Limited viability - Epithelium not intact = corresponds to worst case in vivo scenario

7. Data on candidate microbicides in in vitro models 1) Cell suspension models A) Cell line (GHOST) + single cycle pseudovirus B) DC/T4 co-culture: monocyte-derived dendritic cells + autologous T4 cells + primary replicative virus 2) Models of female genital tract mucosa A) In vitro dual chamber model : DC/T4 + epithelial cells on top B) Ex vivo cervico-vaginal explant = tissue from hysterectomy

8. “Microbicide” Ba-L PV + 30’ Ghost-CD4-CCR5 48h Pre-incubationInfection Production of luciferase 55 TAK77942 T20 Binding gp120:CCR5 Fusion Reverse transcription PMPA UC781 TMC120 L870812 82 2 7 9 CompoundEC 50 (nM) gmeanMechanism of inhibition Binding gp120:CD4 BMS806 5 Integrase Pseudovirus Test = Screening

9. Rescue latent or subliminal infection DC/T4 co-culture with compound 14 days 7 days p24 Ag + HIV-1 infection 2h wash step 30 min ±± or↓ ¤↓ ¤ Cell-free virusCompound ¤ T4 cells Cell-associated virus MO-DC PBMC-PHA/IL-2 activated cells Co-culture model of MO-DC and T4 cells

10. Binding/fusion BMS 5 TAK 779 42 T20 55 140 PMPA 82 TMC-120 2 3 2 UC781 7 111 52 L 870 812 9 183 Pseudovirus DC/T4 co-culture + Ghost-CCR5 + Free HIV + Cell-ass. HIV EC50 (nM) 848 326 > 10,000 4,500 92 RT inhibitors Integrase Inh 125 1,250 Concluding: All compounds active in PV/GHOST (< 100 nM) Binding/fusion inhibitors less active with repicative free HIV inactive with cell-associated HIV Reverse Transc. Inh. very active in all conditions Integrase Inhibitors intermediate profile Summary of cell suspension data

11. In vivoIn vitro Nature Rev 2006, Lederman MM Dual Chamber model

12. 010100 Entry-inhibitors Non-nucleoside reverse transcriptase inhibitors Conc. entry-inhibitor (µg/ml) Conc. NNRTI (nM) % HIV positive cultures CONCLUSION: Binding Inhibitors: rather inactive NNRTI: very active Effect of Binding Inhibitors and NNRTI against Cell-associated HIV in dual chamber model

13. Cervical epithelium (Junction zone) Migratory cells (DC + T cells) Explant model

14. Cervical epithelium Migratory cells Various binding inhibitors UC781 (NNRTI) Conclusion: Binding Inhibitors: active, but less against migratory cells NNRTI: very active, especially against migratory cells (From R Shattock’s group: J Exp Med 2004 and J Virol 2005) Inhibition of cell-free infection in explant model

15. Issues in further development of microbicides Incomplete knowlegde of transmission process: - Cell-free or cell-associated virus ? - Which are the relevant target cells and receptors ? - Role of seminal and cervico-vaginal fluid factors ? - Role of normal vaginal flora/STD and “vaginal practices” ? Avoiding unwanted side-effects: - Enhancing infection by epithelial damage or inflammation - Limiting therapeutic options by induction of resistance

16. Which in vitro test is suitable and predictive? Impossible to say until first succesful human clinical trial, In the mean time:  Use several models reflecting aspects of sexual transmission: e.g. DC and T cells (+ epithelial cells) Explant model Inclusion of seminal and vaginal fluid factors  In addition: - Ensure activity agains cell-free and cell-associated HIV; - Study optimal drug combinations; - Thorough evaluation of toxicity; - Study consequences of possible resistance development.

17. ACKNOWLEDGEMENTS Collaborators: Yven Van Herreweghe; Katty Terrazas; Youssef Gali Jo Michiels; Laetitia Aerts; Leo Heyndrickx Funding EUROPRISE: sponsored this lecture EMPRO: European Microbicides Program ANRS: (France) IWT and FWO: Scientific funds of Flemish government DGOS: Belgian Ministry of Development AmfAR: American Foundation for AIDS Research IPM/TIBOTEC CONRAD ITM institutional support