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

2. 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 Macaque Models of HIV/AIDS

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

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

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

7. Viral DNA is Mainly in Lymphoid and Gut Tissues gag DNA copies/10 6 CCR5 DNA copies in each tissue

8. 1 10 100 1000 10000 100000100000010000000 Week 1 Plasma Viral Load Lymphoid Tissue vDNA (normalized to 10 6 CCR5) 0.996 p < 0.0001 Week 1 Plasma Viremia Correlates with Lymphoid Viral DNA Levels

9. UntreatedART-Treated RT-SHIV gag DNA copies in PBMC

10. Little Viral RNA Detected in Tissues of Suppressed Animals gag RNA copies/10 6 CD4 RNA copies in each tissue

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

12. 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 Conclusions

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 Daria Hazuda Julio Quintero Gilead Sciences Norbert Bischofberger

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

15. R 2 = 0.997 CCR5 Standard Curve R 2 = 0.987 gag Standard Curve RT-SHIV gag DNA vs. Host Cell DNA (CCR5)