Candidate Microbicides: What we can learn from in vitro work

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

2. Vaginal HIV transmission

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

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

6. In Vitro models to test HIV Microbicides - Limited access (HT -) - Risk of damaging epithelium

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 vitrodual chamber model : DC/T4 + epithelial cells on top B) Ex vivo cervico-vaginal explant = tissue from hysterectomy

8. Pre-incubation Mechanism of inhibition Compound EC50 (nM) gmean Binding gp120:CD4 BMS806 5 Binding gp120:CCR5 TAK779 42 Fusion T20 55 Reverse transcription PMPA 82 TMC120 2 UC781 7 L870812 9 Pseudovirus Test = Screening Production of luciferase Infection 30’ 48h + Ghost-CD4-CCR5 Ba-L PV “Microbicide” Integrase

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

10. Summary of cell suspension data EC50 (nM) Pseudovirus DC/T4 co-culture + Ghost-CCR5 + Free HIV + Cell-ass. HIV 5 BMS > 10,000 > 10,000 4,500 326 Binding/fusion 42 848 TAK 779 55 140 T20 82 PMPA 92 125 RT inhibitors 2 3 2 TMC-120 7 111 52 UC781 Integrase Inh 9 183 L 870 812 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

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

12. Conc. entry-inhibitor (µg/ml) 0 10 100 Entry-inhibitors % HIV positive cultures Non-nucleoside reverse transcriptase inhibitors CONCLUSION: Binding Inhibitors: rather inactive NNRTI: very active Conc. NNRTI (nM) Effect of Binding Inhibitors and NNRTI against Cell-associated HIV in dual chamber model

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

14. Inhibition of cell-free infection in explant model Various binding inhibitors UC781 (NNRTI) Cervical epithelium Migratory cells 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)

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