Epigenetic drug Gar1041 in combination with antiretroviral therapy (ART) transiently reduces the proviral DNA reservoir in SIVmac251- infected macaques Andrea Savarino

Objectives To develop an antiretroviral treatment regimen in SIV infected macaques that could be used to test eradication strategies To increase the numbers and classes of effective antiviral drug for use in macaques that mirror treatments in human HIV cases To initiate preliminary studies to determine if virus eradication or disease alteration is possible

Virus Reservoirs Eradication Prevent any virus spread by intensified ART – Target multiple sites of viral inhibition Induce latently infected cells to replicate virus – Histone deacetylase inhibitors Eliminate productive/infected cells – Virus replication/cytotoxicity – Immune response – Passive therapies

Stimulation of HIV-1 LTR-controlled expression of GFP by MS-275 and buthionine sulfoximine (BSO) in a Jurkat cell clone (A1). Savarino, et al., Retrovirology 2009.

Gar1041 ACH2 ctrl 0.6 μM GAR1041 Reactive oxygen species (ROS) induction NF-  B nuclear translocation Gar h Gar h Gar h TNF-  72 h Pos. control (TNF-  1.5 h) p24 induction Gar1041

CD27 Downregulation TNTN T CM T TM T EM Mock 50nM Gar1041

Response of SIVmac251-infected macaques to raltegravir RAL RAL + PMPA + FTC RAL- 100mg BID, oral PMPA - 20mg/kg, SQ FTC – 50mg/kg, SQ RAL RAL + PMPA + FTC Lewis M. Norelli S., et al., Retrovirology 2010.

Treatment of monkeys with ART plus Gar1041 *P < 0.05 **P < 0.01 ***P < P < 0.05

Darunavir (DRV) complexed with HIV-2 proteaseDRV complexed with SIVmac251 protease Barreca ML, Norelli S, and Savarino A, unpublished Kovalevsky et al., 2008

Addition of Ritonavir-boosted Darunavir to the ART/Gar1041 regimen *P < 0.05

iART/Gar1041 (average trend over time: P = ; t-test for regression). iART alone (average trend over time: P = ; t-test for regression). Proviral DNA in PBMC from SIV+ Macaques Following Addition of Ritonavir-boosted Darunavir ART/Gar1041 Regimen.

Stimulation of viral replication by histone deacetylase inhibitor, SAHA in SIVmac251-infected monkeys treated with intensified ART (iART) alone but not in monkeys SIVmac251-infected monkeys treated with iART and Gar1041 iART alone iART plus Gar1041 *P < 0.05 **P < 0.01

Re-appearance of viral loads following treatment suspension *P < 0.05; Gehan-Breslow-Wilcoxon test for survival * * Intensified ART alone Intensified ART plus Gar SAHA cycles 2 SAHA cycles

*P < 0.05; paired Student’s t-test Drug free remission… eventually? iART alone iART plus Gar1041

Drug free remission… eventually? iART alone iART plus Gar1041

Conclusions Our findings suggest that Gar1041 impacts the proviral DNA reservoir in SIVmac251-infected macaques under treatment with ART, likely acting by an entirely novel mechanism (differentiation of long-lived or proliferation- competent memory T cells into short-lived effector-like phenotypes). They also highlight the need for ART intensification when adopting drugs capable of inducing HIV-1 replication. Preliminary studies using oxidative stress inducing agents indicate the utility of the simian model to assess in-vivo the effectiveness of treatments that may stimulate the removal of latently infected cells

Acknowledgments Bioqual Matt Collins Jake Yalley-OgunroUniversity of Rome “Tor Vergata” Jack Greenhouse Enrico Garaci Wendy Wagner ICGEB Istituto Superiore di Sanità, RomeMarina Lusic Sandro Norelli Barbara ChirulloUniversity of Rome, La Sapienza Marco SgarbantiRossella Sgarbanti Andrea SavarinoAnna Teresa Palamara