2014 “Towards an HIV Cure” symposium Melbourne Genetically Characterizing the Role of Cell Proliferation in Maintaining Persistent HIV during Effective.

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

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

Objectives of the Study

Patient 6Treated during chronic infection> 5 years of therapy

PlasmaPre-Therapy - 2004Pre-Therapy - 2005On-Therapy – Time Point 1On-Therapy – Time Point 2

GALT – Effector Memory

GALT – NaiveGALT – Central / Transitional Memory

On-therapy – First Time Point

Peripheral blood – Central / Transitional MemoryPeripheral blood – Effector Memory

Peripheral blood – NaivePeripheral blood – All Memory

On-therapy – Second Time Point

Peripheral blood – Effector Memory

Peripheral blood – NaivePeripheral blood – Central MemoryPeripheral blood – Transitional Memory

GALT – Effector Memory

GALT – NaiveGALT – Central / Transitional Memory

0.005

19 Identical Sequence Expansions2 Identical Sequence Expansions Include Pre-therapy

Identical Sequences Identical sequences during long-term

suppressive therapy>2 genetically identical sequences

Time Point 1 Time Point 2 Time Point 1 Time Point 2Naïve 33% 33% 0% 0%

Memory All 19% N/A N/A N/A

Central/Transitional Memory 18% N/A 27% 0%Central Memory N/A 11% N/A N/A

Transitional Memory N/A 6% N/A N/A

Effector Memory 33% 57% 8% 83%

Cell TypeBlood GALT

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

33% 57%

Patient 8Treated during chronic infection>8 years of therapy

PlasmaPre-Therapy - 2000Pre-Therapy - 2002

GALT – Effector Memory

GALT – NaiveGALT – Central / Transitional Memory

On-therapy – First Time Point

Peripheral blood – Central / Transitional MemoryPeripheral blood – Effector Memory

Peripheral blood – NaivePeripheral blood – All Memory

On-therapy – Second Time Point

Peripheral blood – Effector Memory

Peripheral blood – NaivePeripheral blood – Central MemoryPeripheral blood – Transitional Memory

GALT – Effector Memory

GALT – NaiveGALT – Central / Transitional Memory

Lymph node tissue – Effector Memory

Lymph node tissue – NaiveLymph node tissue – Central MemoryLymph node tissue – Transitional Memory

Identical Sequences

Identical sequences during long-term suppressive therapy

27 Identical Sequence Expansions1 Identical Sequence Expansion Includes Pre-therapy

0.01

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 1 Time Point 2 Time Point 1 Time Point 2 Time Point 1 Time Point 2Naïve 24% 22% N/A N/A N/A 17%

Memory All 38% N/A N/A N/A N/A N/A

Central/Transitional Memory 53% N/A 30% 0% N/A N/A

Central Memory N/A 15% N/A N/A N/A 12%Transitional Memory N/A 35% N/A N/A N/A 26%

Effector Memory 73% 79% 6% 17% N/A 61%

Cell TypeBlood GALT LNT

73% 79% 61%

Time Point 2: EM vs CM > 11-fold p = 0.0003

EM vs TM > 4-fold p = 0.02

61%

Patient 7Treated during chronic infection> 9 years of therapy

PlasmaPre-Therapy - 2002

GALT – Effector Memory

GALT – NaiveGALT – Central / Transitional Memory

On-therapy – First Time Point

Peripheral blood – Central / Transitional MemoryPeripheral blood – Effector Memory

Peripheral blood – NaivePeripheral blood – All Memory

On-therapy – Second Time Point

Peripheral blood – Effector Memory

Peripheral blood – NaivePeripheral blood – Central MemoryPeripheral blood – Transitional Memory

0.005

Identical Sequences

Identical sequences during long-term suppressive therapy

9 Identical Sequence ExpansionsNO Identical Sequence Expansions Include Pre-therapy

Time Point 1 Time Point 2 Time Point 1 Time Point 2Naïve 33% 0% N/A N/A

Memory All 70% N/A N/A N/A

Central/Transitional Memory 0% N/A 50% N/A

Central Memory N/A 57% N/A N/A

Transitional Memory N/A 12% N/A N/A

Effector Memory 82% 92% 19% N/A

Cell TypeBlood GALT

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

82% 92%

0.005

SCOPE2013: Leukapheresis

46

37

100367

78

29

98

0.01

Leuka TCM

Leuka TTM

Leuka TEM

Leuka DN (X5-R6-)

Leuka R6 (X5-R6+)

Leuka X5 (X5+R6-)

Leuka DP (X5+R6+)

On-Therapy

Hypermutants

stop

Viable

Subset Hypermutant (%)

Leuka TCM 50.0 (5/10)

Leuka TTM 11.5 (3/26)

Leuka TEM 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

97

100

13

40

84

42

97

99

0.01

% APD: 1.4%

Subset % Identical sequences

Leuka TCM 50.0 (2/4)

Leuka TTM 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)

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

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.

Acknowledgements

We acknowledge with gratitude the participation of all the patients in this study

Karolinska Institutet L. OdevallS. von Stockenström

VGTIN. Chomont

SAIC/NCIW. Shao

University of California, San FranciscoF. HechtE. SinclairP. BacchettiP. LewisP. HuntM. SomsoukH. HatanoS. DeeksL. EplingM. Kilian T. HoA. TanJ. CusterL. LoebR. HohL. PooleS. PillaiM. Abdel-MohsenA. Raposo

Westmead Millennium InstituteE. LeeB. HienerK. BartonM. LoganT. Cunningham

VRC/NIAIDD. DouekE. Boritz

Rega Institute, BelgiumN. FariaP. Lemey