Eradicating HIV: Understanding the Virus and Developing Effective Treatments

1. Can we eradicate HIV…What do we need to answer the question? Daria HazudaMerck and Co July 2010

2. Why can’t we cure HIV with ARV DrugsWhere is the virus and how is it maintained in the face of “suppressive” therapy? • Residual replication • Sanctuaries; drug penetration • Efficacy, cell type differences • Persistent HIV expression • Replication competent? • Immune disfunction? Latently infected cells Inflammation Homeostatic Proliferation These are not mutually exclusive mechanisms; will multiple approaches be required?

3. Is it the same in all patients? • Time from infection (acute vs chronic) • Initiation of therapy and nadir CD4 • Route of infection • Age • Genetic factors, including • Race • Ethnicity • Gender • ARV regimen • Other, eg., co-infection with HCV, HCMV etc.

4. Why can’t we cure HIV with ARV DrugsWhere is the virus and how is it maintained in the face of “suppressive” therapy? • Residual replication • Sanctuaries; drug penetration • Efficacy, cell type differences • Persistent HIV expression • Replication competent? Latently infected cells Inflammation Homeostatic Proliferation These are not mutually exclusive mechanisms; will multiple approaches be required?

5. Rationale and Goal • Hypothesis • Reactivation of HIV-1 within latent reservoirs in the presence of HAART will lead to elimination of latent reservoirs through a combination of cytopathic viral and immune mechanisms • Goal • Use small molecule(s) to reactivate latent HIV-1 genomes, purge the reservoir and elicit a “sustained virologic response” in the absence of continued antiretroviral therapy Functional Cure

6. Research & Discovery Process Post- Marketing FDA Review 2 1 Phase III n=1000-5000 Phase II n=100-500 Phase I n=20-100 Clinical Trials Increasing biologicalcomplexity 6 5 Compounds 250 Compounds Preclinical 1.5 Drug Discovery 5 5,000 – 10,000 Compounds Years 1DiMasi JA, Hansen RW, Grabowski HG Journal of Health Economics 22 (2003): 151-185

7. How do we test this hypothesis?From the test tube to humans In vitro tools Compound or Target identification Cell-based assays, siRNAs Compounds/drug leads (One or more MOA?) Human studies Biomarkers/clinical surrogates Animal models PD markers; Efficacy Where are we now…

8. HIV LatencyCell Culture Models Integrated LTR-reporter constructs Advantages: LTR is inducible by compounds that activate latent HIV, amenable to siRNA screening Disadvantages: highly reductionist system Chronically infected, inducible cell lines Advantages: complete integrated HIV genome Disadvantages: clonal, each line has a single integration site, some have defective Tat/Tar Retroviral vectors Advantages: GFP reporters allow sorting of population of transduced cells, mixed population Disadvantages: Constructs integrated into heterochromatin; HIV is more likely to integrate into transcribed genes (mixed population

9. HIV LatencyMore Complex Cell Culture Systems • Ex-vivo infected primary resting CD4+ T cells • minimal LTR reporters • LGIT • HIV-1 provirus • Resting CD4+ T cells • Bcl-2-transduced resting CD4+ T cells • Resting CD4+ T cells isolated from HIV+ aviremic patients • Quantification of viral DNA/RNA in memory T cell subsets • Viral outgrowth assays How do they compare? Which is most biologically relevant?

10. Merck High Thoughput Screen Assay for Activators of Latent HIV-1 Gene Expression Uninfected cell b - galactosidase HIV LTR HIV LTR E.coli E.coli lacZ lacZ polyA polyA Uninfected cell + compound b - galactosidase Hela P4/R5 cells 24 hours Add Compounds 24 hours b - Galactosidase Assay

11. Activation of HIV-1 Gene Expression Correlates with HDACi Potency

12. Non-mechanism based screening can identify novel HIV-1 activators ~ 1.5 million compounds (MRL Library) LTR-bGal HTS ~ Confirmed 104 compounds (not known HDACIs) NFAT-BLA Jurkat cell assay ~ 92 compounds that did not activate T-cell HDAC activity assay (novel HDACIs) ~ 83 compounds with potential novel mechanism of Toxicity Chemical attractiveness Further chacterization eg ACH-2, J1.1, primary cells, ex vivo

13. Clinical Samples^ Ex-vivo CD4 T cells* Provirus Jurkat** LBIT Jurkat# LTR-Bgal HeLa# HDAC inhibition pTEFb release NFκB activation Characterizing novel activators +/- Are these meaningful differences or a reflection of the spectrum of relevant biology?

14. Me MT Me MT MT Inhibitor Proposed mechanisms to affect latent proviral HIV-1 expression Adapted from Richman et al, 2009

15. Potential HIV-1 Latency Activation Therapies • Histone deacetylase (HDAC) inhibitors • Class I-selective: SAHA, others (MRL) • Non-selective: Trichostatin A (TSA), valproic acid (VPA) • NF-kB activators • Prostratin, PMA, TNF • Akt/HEXIM-1 modulators • Hexamethylbisacetamide (HMBA) • Histone methyltransferase (HMT) inhibitors • DZNep: targets Ezh2 (trimethylates H3-K27/H4-K20) • Chaetocin: targets su(var)3-9 (methylates H3-K9) • Jak/Stat pathway • IL-7 Lessons from Oncology: Synergy with HDACIs

16. Synergistic reactivation of latent HIV-1 • Synergistic reactivation using combinations of agonists demonstrated in cell lines • Suggested as a potentially more robust approach to reactivate HIV-1 derived from various patients, viral subtypes, or LTR mutants • Low dose may result in diminished AEs in clinic U1 cells J-Lat cells Reuse et al, 2009 Burnett et al, 2010

17. SAHA in combination with MRL HIV “inducing” compounds synergize to activate the HIV LTR Controls Compounds alone Compounds + SAHA A alone B alone A + SAHA B + SAHA C alone D alone C + SAHA D + SAHA J89 Cells: latently infected Jurkat cell line with a single integrated copy of the HIV genome with EGFP as a marker for HIV expression upon stimulation of the LTR. METHODS: J89 cells were incubated with DMSO, L127, L412, L495 or L801 +/- SAHA for 19hrs. GFP expression was measured by flow cytometry. (Archin, et al unpublished)

18. Can Activation Alone “purge” the Reservoir? Latent Cells Immune HDACIs + ??? OR Modulator? Activated cells

19. Can Activation Alone “purge” the Reservoir? Latent Cells Immune HDACIs + ??? plus Modulator? Activated cells ThX Vaccine Immunotoxin, anti-PD1, etc Can you “reset” the immune system without therapy intensification and shutting off persistent antigen production?

20. HIV-1 Latency Pre-clinical In Vivo Models • Animal models are critical for understanding viral persistence and testing novel concepts • Can model HAART in HIV-infected humans (eg, RTIs and InSTIs) • Parameters such as time of infection and HAART initiation can be standardized. • It is possible to extensively evaluate reservoirs in tissues eg, GALT & CNS • Viral rebound as an critical endpoint can be monitored. • Rodent Models • SCID-hu mouse (human transplants of thymus, fetal liver or PBMCs • BLT mouse (human bone marrow, liver, thymus) • More complete systemic reconstitution of all major human hematopoetic lineages including T, B, monocyte/macrophage, dendritic and NK cells. • Macaque Models: SIV, SHIV etc Approaches that delay or decrease viral rebound can provide information for the design of novel and/or combination strategies.

21. RT-SHIV Study: HAART and Induction w/HDACI & PKC activator (Paul Luciw, et al unpublished) RT-SHIV Inoculation Necropsy Necropsy Rebound (16 wks) No treatments 6 wks 32 - 35 wks 8 wks 1wk HAART + Induction HAART (FTC + PMPA + Efavirenz) 2 wks: 5 cycles 6 wks: 1 cycle per wk Weekly/biweekly analysis: plasma viral RNA, CBC, FACS

22. Longitudinal Analysis of Viral Loads During HAART • Low-Level Viremia Persists Despite Effective HAART Fig.3A

23. Tissue vRNA levels are reduced following combination induction treatment and rebound

24. Tissue vDNA levels are reduced following combination induction treatment and rebound

25. No Difference in rebound plasma viremia after discontinuation of HAART and inducers Group 1B = HAART plus induction; Group 3 = HAART only

26. Summary and Outstanding Issues Multiple and perhaps “inter-dependent” processes contribute to the inability to eradicate HIV with ARV therapy Will any one approach be sufficient for eradication? Will the same combination of interventions work for all patients? Various interventions which can address at least some of these issues are being explored including small molecules which may activate latent viral gene expression Activators can manifest differential activity in different cell based assays; Are these differences biologically meaningful or reflect a specturm of biological mechanisms relevant in “non-uniform” systems? HDACIs appear to be the most robust, provide an anchor for combinations? Will activation therapy be sufficient without modulation of the immune response; can the immune response be modulated wiithout blocking pesistent viremia? Evaluating these approaches individually and in combination in well validated animal models will be critical to understand many of these issues What data will provide sufficient evidence to justify clinical evaluation? What clinical surrogates can be used to provide an early signal of efficacy and would be sufficiently robust to trigger therapy interruption?

27. Amy Espeseth Marta Majdan Camil Sayegh Chris Tan Collaborators: David Margolis Una O’Doherty Doug Richman Jeff Lifson Paul Luciw Tom North Warner Greene Eric Verdin Many others Acknowledgements

29. Statistical analysis comparing vRNA and vDNA tissue levels between induction and non-induction groups