Zandrea Ambrose, Ph.D . Division of Infectious Diseases ,

1. “Towards an HIV Cure” Pre-Conference Symposium 20 & 21 July 2012 Viral Tissue Reservoirs Are DeterminedEarly and Little Viral RNA Is Detectedduring Suppression in the Macaque Model Zandrea Ambrose, Ph.D. Division of InfectiousDiseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

2. Macaque Models of HIV/AIDS • infection, pathogenesis and immunity can be similar to that of HIV-infected humans • procedures can be performed more easily and/or ethically than in humans • removal of vital organs at necropsy • acquisition of longitudinal samples • changing/testing novel treatment regimens • model can be controlled in a way that humans cannot • known infecting virus, timing, and route of infection • known treatment adherence, past regimens, and timing of treatments • scheduled blood and tissue sampling

3. gag vif rev tat LTR LTR PR RT IN nef env vpr vpu HIV-1 Does Not Replicate Well in Monkeys HIV-1 gag vif rev tat LTR LTR PR RT IN nef SIV env vpx vpr gag vif rev tat LTR LTR PR RT IN nef RT-SHIV env vpx vpr

4. Study Objectives • Determine which tissues harbor viral DNA that could contribute to the reservoir • How early are these reservoirs established? • Are the levels different after suppressive therapy? • Determine which tissues contain viral RNA • Are multiple sites contributing to viremia in untreated animals? • Is there detectable viral RNA in the tissues of ART-suppressed animals?

5. RT-SHIV Plasma Viremia No Therapy Therapy Therapy 7 6 5 TNV/FTC/EFV PT8272 PT8030 GN19 Viral RNA copies/ml plasma (log) 4 TNV/FTC/EFV/L-870812 GG45 GV08 GV40 3 2 1 0 5 10 15 20 25 30 35 Weeks post-challenge

6. RT-SHIV gag DNA vs. Host Cell DNA (CCR5)

7. Viral DNA is Mainly in Lymphoid and Gut Tissues gag DNA copies/106CCR5 DNA copies in each tissue

8. Week 1 Plasma Viremia Correlates with Lymphoid Viral DNA Levels 10000 0.996 p < 0.0001 1000 100 Lymphoid Tissue vDNA (normalized to 106CCR5) 10 1 10000 100000 1000000 10000000 Week 1 Plasma Viral Load

9. RT-SHIV gag DNA copies in PBMC Untreated ART-Treated

10. Little Viral RNA Detected in Tissues of Suppressed Animals gag RNA copies/106CD4 RNA copies in each tissue

11. PBMC Viral RNA Level is not Reflective of Plasma Viremia or Most Tissues Week 1/2 Week 28/30 Untreated ART-Treated Untreated ART-Treated

12. Conclusions • Viral DNA is detected in many tissues and PBMC of untreated and ART-treated macaques • < 4% of viral DNA from 2-LTR circles • Levels of viral DNA in lymphoid tissues correlated with week 1 plasma viremia • reservoir size is established early after infection • Tissue viral RNA levels were lower and less frequent in ART-treated macaques • No replication-competent virus was detected in LN resting CD4+ T cells of ART-treated animals treated with 4 (< 0.5 IUPM) • Viral RNA levels in PBMC were not consistent with treatment or plasma viremia

13. Acknowledgements University of Pittsburgh Chris Kline Jean Ndjomou Tamera Franks John Mellors AIDS & Cancer Virus Program, NCI Jeff Lifson Jake Estes Mike Piatak, Jr. Jeremy Smedley Vicky Coalter Rebecca Kiser Merck DariaHazuda Julio Quintero Gilead Sciences Norbert Bischofberger

14. Detection of Replication-Competent Virus from Lymph Node CD4+ Lymphocytes

15. RT-SHIV gag DNA vs. Host Cell DNA (CCR5) gag Standard Curve CCR5 Standard Curve R2 = 0.987 R2 = 0.997