2014 “ Towards an HIV Cure ” symposium Melbourne

1. 2014 “Towards an HIV Cure” symposiumMelbourne 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. Identical sequences during long-term suppressive therapy Identical Sequences >2 genetically identical sequences Patient 6 Treated during chronic infection > 5 years of therapy Plasma Pre-Therapy - 2004 Pre-Therapy - 2005 On-Therapy – Time Point 1 On-therapy – First Time Point On-Therapy – Time Point 2 Peripheral blood – Naive Peripheral blood – All Memory Peripheral blood – Central / Transitional Memory Peripheral blood – Effector Memory GALT – Naive GALT – Central / Transitional Memory GALT – Effector Memory 33% 57% On-therapy – Second Time Point Peripheral blood – Naive Peripheral blood – Central Memory Time Point 1: EM vs CTM > 2-fold p = 0.3 Time Point 2: EM vs CM > 11-fold p = 0.001 EM vs TM > 20-fold p < 0.001 Peripheral blood – Transitional Memory Peripheral blood – Effector Memory GALT – Naive GALT – Central / Transitional Memory GALT – Effector Memory 2 Identical Sequence Expansions Include Pre-therapy 19 Identical Sequence Expansions

6. Identical sequences during long-term suppressive therapy Identical Sequences Patient 8 Treated during chronic infection >8 years of therapy Plasma Pre-Therapy - 2000 Pre-Therapy - 2002 On-therapy – First Time Point Peripheral blood – Naive Peripheral blood – All Memory Peripheral blood – Central / Transitional Memory Peripheral blood – Effector Memory GALT – Naive GALT – Central / Transitional Memory GALT – Effector Memory 73% 79% 61% 61% On-therapy – Second Time Point Lymph node tissue – Naive Peripheral blood – Naive Time Point 1: EM vs CTM > 2-fold p = 0.2 Time Point 2: EM vs CM > 20-fold p = 0.0001 EM vs TM 7-fold p = 0.01 Time Point 2: EM vs CM > 11-fold p = 0.0003 EM vs TM > 4-fold p = 0.02 Lymph node tissue – Central Memory Peripheral blood – Central Memory Lymph node tissue – Transitional Memory Peripheral blood – Transitional Memory Lymph node tissue – Effector Memory Peripheral blood – Effector Memory GALT – Naive GALT – Central / Transitional Memory GALT – Effector Memory 27 Identical Sequence Expansions 1 Identical Sequence Expansion Includes Pre-therapy

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

8. SCOPE2013: Leukapheresis Treated during chronic infection > 15 years of therapy Viable Hypermutants % APD: 1.4% stop On-Therapy  Leuka DN (X5-R6-)  Leuka TCM  Leuka TTM  Leuka R6 (X5-R6+)  Leuka TEM  Leuka X5 (X5+R6-)  Leuka DP (X5+R6+)

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 Karolinska Institutet L. Odevall S. von Stockenström 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 SAIC/NCI W. Shao Westmead Millennium Institute E. Lee B. Hiener K. Barton M. Logan T. Cunningham VGTI N. Chomont VRC/NIAID D. Douek E. Boritz Rega Institute, Belgium N. Faria P. Lemey We acknowledge with gratitude the participation of all the patients in this study