1. 2014 “Towards an HIV Cure” symposium Melbourne Genetically Characterizing the Role of Cell Proliferation in Maintaining Persistent HIV during Effective HIV Therapy Sarah Palmer Westmead Millennium Institute and University of Sydney

2. Background A diverse repertoire of naive and memory T cells is maintained to defend against new and previously encountered pathogens The level of peripheral T cells is closely regulated and is maintained by cellular proliferation and homeostatic proliferation which takes place within the T cell area of the spleen and lymph nodes However, T cell proliferation, differentiation and activation have poorly defined effects on the latent HIV reservoir during potent cART

3. Objectives of the Study To genetically characterize the role of cell proliferation in maintaining persistent HIV: examine the distribution of identical HIV-1 intracellular sequences in resting memory T cells: - peripheral blood -GALT -lymph node tissue with the aim to define the role of cell proliferation as a cause of persistence in different memory T cell subsets during long-term suppressive therapy

5. Patient 6 Treated during chronic infection > 5 years of therapy Plasma Pre-Therapy - 2004 Pre-Therapy - 2005 On-Therapy – Time Point 1 On-Therapy – Time Point 2 GALT – Effector Memory GALT – Naive GALT – Central / Transitional Memory On-therapy – First Time Point Peripheral blood – Central / Transitional Memory Peripheral blood – Effector Memory Peripheral blood – Naive Peripheral blood – All Memory On-therapy – Second Time Point Peripheral blood – Effector Memory Peripheral blood – Naive Peripheral blood – Central Memory Peripheral blood – Transitional Memory GALT – Effector Memory GALT – Naive GALT – Central / Transitional Memory 19 Identical Sequence Expansions 2 Identical Sequence Expansions Include Pre-therapy Identical Sequences Identical sequences during long-term suppressive therapy >2 genetically identical sequences Time Point 1:EM vs CTM> 2-foldp = 0.3 Time Point 2:EM vs CM> 11-foldp = 0.001 EM vs TM> 20-foldp < 0.001 33% 57%

6. Patient 8 Treated during chronic infection >8 years of therapy Plasma Pre-Therapy - 2000 Pre-Therapy - 2002 GALT – Effector Memory GALT – Naive GALT – Central / Transitional Memory On-therapy – First Time Point Peripheral blood – Central / Transitional Memory Peripheral blood – Effector Memory Peripheral blood – Naive Peripheral blood – All Memory On-therapy – Second Time Point Peripheral blood – Effector Memory Peripheral blood – Naive Peripheral blood – Central Memory Peripheral blood – Transitional Memory GALT – Effector Memory GALT – Naive GALT – Central / Transitional Memory Lymph node tissue – Effector Memory Lymph node tissue – Naive Lymph node tissue – Central Memory Lymph node tissue – Transitional Memory Identical Sequences Identical sequences during long-term suppressive therapy 27 Identical Sequence Expansions 1 Identical Sequence Expansion Includes Pre-therapy Time Point 1:EM vs CTM> 2-foldp = 0.2 Time Point 2:EM vs CM> 20-foldp = 0.0001 EM vs TM7-foldp = 0.01 73%79%61% Time Point 2:EM vs CM> 11-foldp = 0.0003 EM vs TM> 4-foldp = 0.02 61%

7. Patient 7 Treated during chronic infection > 9 years of therapy Plasma Pre-Therapy - 2002 GALT – Effector Memory GALT – Naive GALT – Central / Transitional Memory On-therapy – First Time Point Peripheral blood – Central / Transitional Memory Peripheral blood – Effector Memory Peripheral blood – Naive Peripheral blood – All Memory On-therapy – Second Time Point Peripheral blood – Effector Memory Peripheral blood – Naive Peripheral blood – Central Memory Peripheral blood – Transitional Memory Identical Sequences Identical sequences during long-term suppressive therapy 9 Identical Sequence Expansions NO Identical Sequence Expansions Include Pre-therapy Time Point 1:EM vs CTM> 30-foldp < 0.0001 Time Point 2:EM vs CM9-foldp = 0.002 EM vs TM> 86-foldp < 0.0001 82%92%

8. SCOPE2013: Leukapheresis  Leuka T CM  Leuka T TM Leuka T EM  Leuka DN (X5-R6-)  Leuka R6 (X5-R6+) Leuka X5 (X5+R6-)  Leuka DP (X5+R6+) On-Therapy Hypermutants stop Viable SubsetHypermutant (%) Leuka T CM 50.0 (5/10) Leuka T TM 11.5 (3/26) Leuka T EM 100 (24/24) Leuka DN (X5-R6-)90.9 (20/22) Leuka X5-R6+90.5 (19/21) Leuka X5+R6-0.0 Leuka DP (X5+R6+)0.0 Treated during chronic infection > 15 years of therapy % APD: 1.4% Subset % Identical sequences Leuka T CM 50.0 (2/4) Leuka T TM 52.2 (12/23) Leuka DN (X5-R6-)0.0 (0/2) Leuka R6 (X5-R6+)0.0 (0/2) Leuka X5 (X5+R6-)75.5 (3/4) Leuka DP (X5+R6+)50.0 (3/6)

9. Cellular Restriction Factors Legend: SLFN11: inhibits viral protein synthesis Tetherin: blocks viral release APOBEC3: Hypermutation; lethal mutations in viral DNA MX2: blocks nuclear import of subviral complexes; blocks integration

10. Conclusions  A large percentage of intracellular sequences, not represented in pre-therapy plasma, are clonal in nature.  These identical sequences are enriched during therapy and in more differentiated cells ie: effector memory.  This suggests that HIV persistence during effective therapy is driven in large part by the proliferation, differentiation and expansion of cell populations with sustained and durable regenerative potential.

11. Acknowledgements We acknowledge with gratitude the participation of all the patients in this study Karolinska Institutet L. Odevall S. von Stockenström VGTI N. Chomont SAIC/NCI W. Shao University of California, San Francisco F. Hecht E. Sinclair P. Bacchetti P. Lewis P. Hunt M. Somsouk H. Hatano S. Deeks L. Epling M. Kilian T. Ho A. Tan J. Custer L. Loeb R. Hoh L. Poole S. Pillai M. Abdel-Mohsen A. Raposo Westmead Millennium Institute E. Lee B. Hiener K. Barton M. Logan T. Cunningham VRC/NIAID D. Douek E. Boritz Rega Institute, Belgium N. Faria P. Lemey