Towards an ART-less Future The Challenge of a Cure for HIV Infection David M. Margolis, MD Professor of Medicine, Microbiology & Immunology, Epidemiology

Lohse, N. et. al. Ann Intern Med 2007;146:87-95 Antiretroviral therapy: >3 million years of life saved Predicted Survival if HIV+ at age 25 years

More than two decades of pandemic HIV: what is to be done in the coming decades? Prevent disease in the infected Prevent infection Eradicate infection

Prevent Disease in the Infected Challenges of Long-term Antiretroviral Therapy Drug Resistance Cost Adherence Long-term Toxicities For every patient that starts therapy, 3 to 4 new infections occur

Prevent infection Education and behavior modification Condoms, and other barrier methods Treatment/prevention of drug/alcohol abuse Clean syringes (i.e. needle exchange programs) Interruption of mother-to-child transmission Circumcision HIV/STI Testing Antiretroviral treatment as prevention Pre-exposure prophylaxis (PrEP) Topical microbicides Vaccination

Pierson et al 2001 Eradicate HIV Infection Today: Persistent HIV Infection despite ART

Maldarelli et al. PLoS Path copies 10 copies 1 copy Low-level viremia Dornadula JAMA 1999 Palmer et al. J Clin Micro 2003: Single-copy assay detected in ca. 75% ART-suppressed patients Level correlates with peak viremia

5 Patients on ART with NNRTI- or PI- based regimens including 2 NRTI stable HIV-1 RNA 1 yr Single copy assay >1 c/mL at entry 30-day drug intensification study with raltegravir 400 mg twice daily before intensification median 1.9 c/mL; during raltegravir 3.2 c/mL not different, p = 0.72 Pre-RAL 0.04 l P=0.69P=0.38 HIV-1 RNA (log 10 copies/mL) No Decrease in Residual Viremia during Raltegravir Intensification in Patients on Standard ART Jones, et al. PNAS 2009 RALPost-RAL Unaffected by intensification with PI, NNRTI, RAL, Enf

randomized 65 patients with 1 year intensify with RAL (n=44), or continue ART (n = 21) for 48 weeks Reported measures at weeks 0, 2, 4, and 12 of: episomal (circular) HIV DNA Integrated HIV DNA Total proviral DNA SCA results not yet reported Overall, transient, significant increase (p = ) in episomal HIV-1 DNA at week 2 (but return to baseline at week 4) Transient Increase in Episomal Viral cDNA following Raltegravir Intensification of a Stable HAART Regimen M Buzon, J Llibre, J Gatell, P Domingo, R Paredes, S Palmer, M Sharkey, M Stevenson, B Clotet and Javier Martinez-Picado CROI 2009; Abstr. 423a

Persistent virion expression without complete rounds of replication How is this productive pool of cells maintained? Inadequate ART in compartments? Homeostatic Proliferation/Mitosis? Long-lived cells, resistant to apoptosis? Direct cell-to-cell infection in tissue? Rudnicka J Virol 2009

Pierson et al 2001 Rare, persistent proviral genomes Persistent HIV Infection despite ART

Maintenance Activation Targeting latency in resting CD4+ T cells HIV-infected Resting CD4+ T cells K in K out ART K out Establishment Derepression Fraser AIDS 2002

Reported Mechanisms of Latency: which can be modulated? Integration site Host gene transcriptional read-through Resting state of memory T cells, low NFAT, NF-kB IBIB HMBA Prostratin

Reported Mechanism of Latency: which can be modulated? Integration site Host gene transcriptional read-through Resting state of memory T cells, low NFAT, NF-kB Host miRNA: HIV RNA translational effect, HIV LTR heterochromatin effect Resting cell deficient in RNA export factor: PTB

Reported Mechanism of Latency: which can be modulated? Integration site Host gene transcriptional read-through Resting state of memory T cells, low NFAT, NF-kB Resting cell deficient in RNA export factor: PTB Host miRNA: HIV RNA translational effect, HIV LTR heterochromatin effect Epigenetics of viral promoter: acetylation, methylation, etc.

deacetylated HIV lives within chromatin “Closed” Nucleosome Gene Expression Repressed “Open” Histones Gene Expression Active acetylated

Nuc-1 Nuc-2 Nuc-0 LTR “Opening” of Chromatin is Required for HIV Expression Nuc-1 Nuc-2 Nuc-0 Van Lint, Emiliani, Ott, Verdin 1996 Sheridan, Mayall, Verdin, Jones 1997 El Kharroubi, Piras, Zensen, Martin 1998 Histone acetyl- transferase NF-  B, Sp1

Nuc-1 Nuc-2 Nuc-0 LTR Margolis et al., J Virol 1994 Romerio et al., J Virol 1997 Coull et al., J Virol 2000 He & Margolis, MCB 2002 Ylisastigui et al. JID 2002 Histone deacetylases can shut down HIV expression LSF YY1 HDAC1 Nuc-1

deacetylated HIV lives within chromatin “Closed” Nucleosome Gene Expression Repressed “Open” Histones Gene Expression Active acetylated

Nuc-1 Redundancy in HDAC1 action at the LTR NF-  B, Sp1 Nuc-2 Nuc-0 HDAC1 recruitment by YY1/LSF Romerio et al., J Virol 1997 Coull et al., J Virol 2000 He & Margolis, MCB 2002 Coull et al. J Virol 2002 Ylisastigui et al. JID 2002 LTR HDAC1 recruitment by p50 of NF-kB Williams et al HDAC1 recruitment by Sp1/c-Myc Jiang et al HDAC1 recruitment by CBF-1 Tyagi & Karn 2007 HDAC1 recruitment by Ap4 Imai & Okamoto 2006

Expected decay t 1/2 =44 mo.     < 9 31 Effect of VPA and Enfuvirtide on Resting CD4+ cell infection (per billion) Lehrman et al. Lancet 2005 Stability of the Latent Reservoir for HIV-1 in Patients Receiving Valproic Acid. Siliciano et al., J Infectious Diseases 2007;195: Prolonged valproic acid treatment does not reduce the size of latent HIV reservoir. Sagot-Lerolle, N. et al. AIDS 2008; 22:

* non-consecutive episodes of viremia >50 copies during study † 6 to 10 determinations from screening visit to week 24 end-of-study visit § Only two assays evaluable in patient 10 and three in patient 11 Infected resting CD4+ T cells per billion Valproic acid without intensified ART has a limited effect on resting CD4 T cell infection. Archin et al. AIDS 2008

Why are the clinical effects of HDAC inhibition not more impressive? Why is persistent HIV so hard to treat? Other viral reservoirs with persistent viral expression More potent HDAC inhibitors may be required More than one mechanism maintains latency; more than one mechanism must be attacked

Which of the 11 HDACs matter in patient’s resting CD4+ T cells Class IClass IIClass IV Keedy J Virol 2009

N C Rest Act HDAC8 N C Rest Act HDAC1 N C Rest Act HDAC2 N C Rest Act HDAC3 Lamin B1: GAPDH: N C Rest Act HDAC11 Lamin B1: GAPDH: Class IClass IV N C Rest Act HDAC4 N C Rest Act HDAC5 N C Rest Act HDAC6 Lamin B1: GAPDH: N C Rest Act HDAC7 N C Rest Act HDAC9 N C Rest Act HDAC10 Class II Nuclear localization of HDACs 1, 2 & 3 and 4, 6 & 7 in patient’s resting CD4+ T cells Keedy J Virol 2009

α-AcH3 α-HDAC1 α-HDAC2 α-HDAC3 IgG 10% Input IP: TSA: - + IP: 10% Input IgG α-AcH4 α-HDAC4 IP: 10% Input IgG α-AcH4 α-HDAC6 IP: 10% Input IgG α-AcH4 α-HDAC7 HDACs 1, 2 & 3 found at the HIV LTR HDACs 4, 6 & 7 not found Keedy J Virol 2009

Antibodies Used: CD4 purification CD8, CD14, CD16 CD19, CD56 Glycophorin A Resting cell cocktail CD41, CD25, HLA-DR Mix cells with Antibody-magnetic bead cocktail Magnet million resting CD4+ cells 4 billion lymphocytes from aviremic patient on HAART Measuring resting CD4+ T cell infection (“latency”) Resting CD4 + Cell Viral Outgrowth Assay: Purified Resting cells Incubated with Integrase Inhibitor & RT Inhibitor Viral Induction Outgrowth assay PHA/IL2/allo IL2 Test drug Single donor, R5-high, CD8-depleted PBMC targets twice a week; p24 day 17, confirm day million cells/well 0.5 million cells/well 0.1 million cells/well Assay variance: 0.3 log Range: 25 cells/million to 9 cells/billion

Infected units per billion resting CD4+ T cells PHA Class II inhibitor Infected units per billion resting CD4+ T cells PHA Class I inhibitor Infected units per billion resting CD4+ T cells PHAVPA global HDAC inhibitor Infected units per billion resting CD4+ T cells PHA Class II inhibitor Limit of detection Archin AIDS 2009

Infected units per billion resting CD4+ T cells PHA HDAC 1, 2, 3 inhibitor Archin et al AIDS 2009 Class I inhibitor HDAC 1, 2 inhibitor HDAC 1, 2, 3 inhibitor T Cell model system Patient’s cell assay

Vorinostat: Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor with nanomolar potency licensed for the treatment of cutaneous T cell lymphoma Inhibits HDACs 1, 2, 3, and 8 (class I) and HDAC 6 (class II) Archin ARHR 2009 Contreras JBC 2009

Latent HIV is constrained by more than one mechanism; more than one mechanism must be attacked

Blazkova PLoS Path 2009 Kauder PLoS Path 2009

Modeling ART in HIV infected BLT mice P Denton, JV Garcia -Martinez

Prevent Infection “Test and Treat” Start ART Target residual viremia (immunotherapy?) Anti-latency therapy …..eradication or remission Future Challenges

What do we need to attack persistent HIV infection? Persistence Studies of the basic biology of latency New and better measures of persistence in patients Studies across all models: cell lines, primary cell models, patient cells, animal models, and patients Approaches to persistent viremia –Not all patients have it: why? –If intensified ART has no effect, can we augment the immune response or directly kill productive cells? Approaches to persistent provirus –HDAC inhibitors, other epigenetic drugs –Combination therapies to induce expression and cell death –Oncology paradigm and paradox

Nancy ArchinKara Keedy Manzoor CheemaDaniel Parker Robbie SackmannKriston Barton Shailesh Choudhary Mike Cohen Joe Eron & ACTU Linh Ngo, JoAnn Kuruc, Alyssa Sugarbaker, Jenny Scepanski UNC Blood Bank staff CFAR labs: Ron Swanstrom, Angela Kashuba John Schmitz, Susan Fiscus, Julie Nelson & CFAR labs And special thanks to our study volunteers Daria Hazuda Ron Bosch Anne Weigand, John Coffin Paul Denton, Victor Garcia

siRNAs targeting HDAC2 or 3 Activate HIV Gene Expression *** Keedy J Virol 2009

siRNAs targeting HDAC2 Induce HIV Outgrowth Keedy preliminary data

Archin 2009