Transcript Targeting the Reservoir: Towards Therapies for Persistent HIV and

The Challenge of a Cure for HIV Infection
Towards an ART-less Future
David M. Margolis, MD
Professor of Medicine, Microbiology & Immunology, Epidemiology
Antiretroviral therapy: >3 million years of life saved
Predicted Survival if HIV+ at age 25 years
Lohse, N. et. al. Ann Intern Med 2007;146:87-95
More than two decades of
pandemic HIV:
what is to be done in the coming decades?
• Prevent disease in the infected
• Prevent infection
• Eradicate infection
Prevent Disease in the Infected
Challenges of Long-term Antiretroviral Therapy
•
•
•
•
Drug Resistance
Cost
Adherence
Long-term Toxicities
For every patient that starts therapy,
3 to 4 new infections occur
Prevent infection
Education and behavior modification
Condoms, and other barrier methods
Treatment/prevention of drug/alcohol abuse
Clean syringes (i.e. needle exchange programs)
Interruption of mother-to-child transmission
Circumcision
HIV/STI Testing
Antiretroviral treatment as prevention
Pre-exposure prophylaxis (PrEP)
Topical microbicides
Vaccination
Eradicate HIV Infection
Today: Persistent HIV Infection despite ART
Pierson et al 2001
Low-level viremia
Dornadula JAMA 1999
Palmer et al. J Clin Micro 2003:
•
•
Single-copy assay
detected in ca. 75% ART-suppressed patients
Level correlates with peak viremia
100 copies
10 copies
1 copy
Maldarelli et al. PLoS Path 2007
No Decrease in Residual Viremia during Raltegravir Intensification
in Patients on Standard ART Jones, et al. PNAS 2009
P=0.69
P=0.38
2
HIV-1 RNA (log10 copies/mL)
• 5 Patients on ART with NNRTI- or
PI- based regimens including 2
NRTI
• stable HIV-1 RNA <50 c/mL for >1
yr
• Single copy assay >1 c/mL at
entry
• 30-day drug intensification study
with raltegravir 400 mg twice daily
• before intensification median 1.9
c/mL; during raltegravir 3.2 c/mL
not different, p = 0.72
1
0
0.04 l
0.14
0.04
-1
Pre-RAL
RAL
Post-RAL
Unaffected by intensification with PI, NNRTI, RAL, Enf
Transient Increase in Episomal Viral cDNA following Raltegravir
Intensification of a Stable HAART Regimen
M Buzon, J Llibre, J Gatell, P Domingo, R Paredes, S Palmer, M Sharkey, M Stevenson, B Clotet and
Javier Martinez-Picado
• randomized 65 patients with <50 c/mL for > 1 year
• intensify with RAL (n=44), or continue ART (n = 21) for 48 weeks
• Reported measures at weeks 0, 2, 4, and 12 of:
episomal (circular) HIV DNA
Integrated HIV DNA
Total proviral DNA
• SCA results not yet reported
• Overall, transient, significant increase (p = 0.0391) in episomal HIV-1
DNA at week 2 (but return to baseline at week 4)
CROI 2009; Abstr. 423a
Persistent virion expression without
complete rounds of replication
How is this productive pool of cells maintained?
Inadequate ART in compartments?
Homeostatic Proliferation/Mitosis?
Long-lived cells, resistant to apoptosis?
Direct cell-to-cell infection in tissue?
Rudnicka J Virol 2009
Persistent HIV Infection despite ART
Rare, persistent proviral genomes
Pierson et al 2001
Targeting latency in resting CD4+ T cells
Establishment
ART
K in
Maintenance
Derepression
Activation
HIV-infected
Resting
CD4+ T cells
K
K out
out
Fraser AIDS 2002
Reported Mechanisms of Latency:
which can be modulated?
• Integration site
• Host gene transcriptional read-through
• Resting state of memory T cells, low NFAT, NF-kB
IB
Reported Mechanism of Latency:
which can be modulated?
•
•
•
•
•
Integration site
Host gene transcriptional read-through
Resting state of memory T cells, low NFAT, NF-kB
Resting cell deficient in RNA export factor: PTB
Host miRNA: HIV RNA translational effect, HIV
LTR heterochromatin effect
Reported Mechanism of Latency:
which can be modulated?
•
•
•
•
•
Integration site
Host gene transcriptional read-through
Resting state of memory T cells, low NFAT, NF-kB
Resting cell deficient in RNA export factor: PTB
Host miRNA: HIV RNA translational effect, HIV LTR
heterochromatin effect
• Epigenetics of viral promoter: acetylation, methylation,
etc.
HIV
lives
within
chromatin
“Closed” Nucleosome
Gene Expression Repressed
deacetylated
acetylated
“Open” Histones
Gene Expression Active
“Opening” of Chromatin is Required for
HIV Expression
LTR
Histone
acetyltransferase
NF-B, Sp1
Van Lint, Emiliani, Ott, Verdin 1996
Sheridan, Mayall, Verdin, Jones 1997
El Kharroubi, Piras, Zensen, Martin 1998
Histone
deacetylases
can shut down
HIV expression
LTR
HDAC1
YY1
LSF
LSF
Margolis et al., J Virol 1994
Romerio et al., J Virol 1997
Coull et al., J Virol 2000
He & Margolis, MCB 2002
Ylisastigui et al. JID 2002
HIV
lives
within
chromatin
“Closed” Nucleosome
Gene Expression Repressed
deacetylated
acetylated
“Open” Histones
Gene Expression Active
Redundancy in HDAC1 action at the LTR
HDAC1 recruitment by YY1/LSF
Romerio et al., J Virol 1997
Coull et al., J Virol 2000
He & Margolis, MCB 2002
Coull et al. J Virol 2002
Ylisastigui et al. JID 2002
HDAC1 recruitment
by Ap4
Imai & Okamoto 2006
LTR
NF-B, Sp1
HDAC1 recruitment
by p50 of NF-kB
Williams et al. 2006
HDAC1 recruitment
by CBF-1
Tyagi & Karn 2007
HDAC1 recruitment
by Sp1/c-Myc
Jiang et al. 2007
Effect of VPA and Enfuvirtide on Resting CD4+ cell infection (per billion)
1000
100
Expected decay
t1/2=44 mo.
49
IUPB
Resting
CD4+
T cells

250


35
100
Patient 1
Patient 3

81
Stability
of the Latent Reservoir for HIV-1 in Patients Receiving
10
2
4
0
2
4
6
6
Valproic
Acid.
Siliciano
et al.,
J Infectious 0Diseases
2007;195:833836
100
136

not
Prolonged
valproic acid treatment does
reduce the size of latent
100

IUPBHIV reservoir. Sagot-Lerolle, N. et al. AIDS 2008; 22:1125-1129
57
Resting
CD4+
T cells

31
10
Patient 2
0

<9
2
4
6
Months on protocol therapy
Patient 4
10
0
2
4
Months on protocol therapy
6
Lehrman et al. Lancet 2005
Valproic acid without intensified ART has a limited effect on
resting CD4 T cell infection. Archin et al. AIDS 2008
Infected resting CD4+ T cells per billion
Patient
1
2
3
4
5
6
7
8
9
10
11
ART
IUPB
187
52
70
183
112
54
531
17951
4890
696
57
ART and
VPA IUPB
90
22
22
3
120
75
625
9438
3300
1253
83
>50% depletion
IUPB on VPA
Yes
Yes
Yes
Yes
No
No
No
No*
No*
No
No
1-copy assay range
median
<1 - 1
<1
<1 - 3
1
<1 - 5
2
n.d.
n.d.
n.d.
--<1 - 1
<1
1-7
3
8 - 77
25
24 - 87
37
<1¤
--<1 Š 1¤
---
* non-consecutive episodes of viremia >50 copies during study
† 6 to 10 determinations from screening visit to week 24 end-of-study visit
§ Only two assays evaluable in patient 10 and three in patient 11
Why are the clinical effects of HDAC inhibition
not more impressive?
Why is persistent HIV so hard to treat?
•Other viral reservoirs with persistent viral expression
•More potent HDAC inhibitors may be required
•More than one mechanism maintains latency; more than one
mechanism must be attacked
Which of the 11 HDACs matter in
patient’s resting CD4+ T cells
Class I
Class II
Class IV
Keedy J Virol 2009
Nuclear localization of HDACs 1, 2 & 3 and 4, 6 & 7
in patient’s resting CD4+ T cells
Class I
Class IV
HDAC1
HDAC2
HDAC3
HDAC8
HDAC11
Rest Act
N C N C
Rest Act
N C N C
Rest Act
N C N C
Rest Act
N C N C
Rest Act
N C N C
Lamin B1:
Lamin B1:
GAPDH:
GAPDH:
Class II
HDAC4
HDAC5
HDAC6
HDAC7
HDAC9
HDAC10
Rest Act
N C N C
Rest Act
N C N C
Rest Act
N C N C
Rest Act
N C N C
Rest Act
N C N C
Rest Act
N C N C
Lamin B1:
GAPDH:
Keedy J Virol 2009
IP:
HDACs 1, 2 & 3
found at
the HIV LTR
TSA:
-
+
10% Input
10%
Input
IP:
+
IgG
-
+
α-AcH4
-
+
α-HDAC4
10% Input
IP:
IgG
α-AcH4
α-HDAC6
10% Input
IP:
IgG
α-AcH4
α-HDAC7
HDACs
4, 6 & 7
not
found
Keedy J Virol 2009
Measuring resting CD4+ T cell infection (“latency”)
Resting CD4+ Cell Viral Outgrowth Assay:
Assay
variance:
4 billion
0.3 log
lymphocytes
Range:
from
25 cells/million
patient
toaviremic
9 cells/billion
on HAART
Mix cells with
Antibody-magnetic bead
cocktail
Single donor, R5-high,
CD8-depleted PBMC
Magnet
targets twice
a week;
p24 day 17, confirm day 19
Magnet
Purified Resting cells
Incubated with Integrase
Inhibitor & RT Inhibitor
Antibodies Used:
PHA/IL2/allo
Test drug
CD4
purification
2.5
million
cells/well
CD8, CD14, CD16
2.5CD19,
million
cells/well
CD56
A
0.5 Glycophorin
million cells/well
0.1 million
Restingcells/well
cell cocktail
CD41, CD25, HLA-DR
Viral Induction
IL2
200-1000
million
resting
CD4+ cells
Outgrowth assay
Infected units per billion resting
CD4+ T cells
10000
1000
100
10
Infected units per billion resting
CD4+ T cells
Infected units per billion resting
CD4+ T cells
100000
10000
1000
100
10
Limit of
detection
PHA
PHA
Class II
inhibitor
Infected units per billion resting
CD4+ T cells
Archin AIDS 2009
10000
1000
100
10
VPA
global HDAC
inhibitor
PHA
PHA
Class I
inhibitor
1000
100
10
Class II
inhibitor
HDAC
1, 2, 3
inhibitor
50
40
30
HDAC 1, 2
inhibitor
20
10
0
MRK1- 1µM
MRK12- 20µM MRK13- 200nM
T Cell model system
Patient’s cell assay
Infected units per billion
resting CD4+ T cells
Percent (%) GFP positive cells above
unttreated control
60
Class I
inhibitor
1000
100
10
PHA
Archin et al AIDS 2009
HDAC
1, 2, 3
inhibitor
Vorinostat:
Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor with
nanomolar potency licensed for the treatment of cutaneous T cell lymphoma
Inhibits HDACs 1, 2, 3, and 8 (class I)
and HDAC 6 (class II)
Archin ARHR 2009
Contreras JBC 2009
Latent HIV is constrained by more than one mechanism;
more than one mechanism must be attacked
Blazkova PLoS Path 2009
Kauder PLoS Path 2009
Modeling ART in HIV infected BLT
mice
P Denton,
JV Garcia
-Martinez
Future Challenges
Start ART
Target residual viremia
Prevent Infection
“Test and Treat”
(immunotherapy?)
…..eradication
or remission
Anti-latency
therapy
What do we need to attack
persistent HIV infection?
• Studies of the basic biology of latency
• New and better measures of persistence in patients
• Studies across all models: cell lines, primary cell
models, patient cells, animal models, and patients
• Approaches to persistent viremia
–Not all patients have it: why?
–If intensified ART has no effect, can we augment the immune
response or directly kill productive cells?
• Approaches to persistent provirus
–HDAC inhibitors, other epigenetic drugs
–Combination therapies to induce expression and cell death
–Oncology paradigm and paradox
Persistence
Nancy Archin
Manzoor Cheema
Robbie Sackmann
Shailesh Choudhary
Kara Keedy
Daniel Parker
Kriston Barton
Daria Hazuda
Ron Bosch
Anne Weigand, John Coffin
Paul Denton, Victor Garcia
• Mike Cohen
• Joe Eron & ACTU
• Linh Ngo, JoAnn Kuruc, Alyssa Sugarbaker, Jenny
Scepanski
• UNC Blood Bank staff
• CFAR labs: Ron Swanstrom, Angela Kashuba
John Schmitz, Susan Fiscus, Julie Nelson
& CFAR labs
And special thanks to our study volunteers
siRNAs targeting HDAC2 or 3
Activate HIV Gene Expression
***
Keedy J Virol 2009
siRNAs targeting HDAC2
Induce HIV Outgrowth
Keedy preliminary data
Archin 2009