Targeting ‘Residual HIV’ In Its
Reservoirs: Where We Are And
Where Do We Go?
HIV Reservoirs Workshop
Vienna, Austria
July 17,2010
Frank Maldarelli, M.D., Ph.D.
HIV Drug Resistance Program
NCI–Frederick
HIV Response to Antiretroviral Therapy
ARV
105
104
HIV-1
RNA
103
102
Detection limit
101
0
8
4
Time (weeks)
16
Decay Kinetics of Viral Infected Cells
108
107
Activated
Lymphocyte
HIV-1
Infected Cells
106
Longer lived
cells
Macrophage?
105
Detection limit
104
1
Longer
14
Half life of infected cells (days)
Identifying the source of HIV viremia during
suppressive antiretroviral therapy is essential to
eradication
Active replication cycles
Stable reservoirs
X
Infected cell
Uninfected cell
HIV production from
active replication is
blocked by ARV therapy
HIV production from
reservoirs is NOT
blocked by ARV therapy
IMPROVED ARV NEEDED
NEW STRATEGIES
NEEDED
Quantitative Measures For Clinical
Studies of HIV Reservoirs
• HIV nucleic acid analysis
• HIV population genetics
Quantitative Measures For Clinical
Studies of HIV Reservoirs
• HIV nucleic acid analysis
• HIV population genetics
Single Copy Quantitation of HIV-1
Viremia
•
•
•
•
•
Real time PCR assay
Linear quantitation 1 - 106 copies HIV-1 RNA
Limit of detection 0.2 copies /ml plasma
Does NOT measure a biological activity
Assay is NOT FDA approved
Superior
Efficacy
ofin
Lopinavir/ritonavir
Selected
130 patients
(67
NFV, 63 LPV/r)
Does
a difference
antiviral
potency
Remainedover
copies/ml
wk 24
Nelfinavir
impact<50
viremia
onfollowing
therapy?
Abbott 98-863 Study
lopinavir/ritonavir
Percent maintaining
virologic response
21% Failure
44% Failure
nelfinavir
p<0.001, Cox proportional hazards model
Week
Walmsley, S. N. Engl. J. Med., 2002
Distribution Rank (Percentile)
Viremia on Therapy is Independent of Regimen
100
80
60
40
Median
0.53
0.48
0.35
lopinavir/ritonavir
nelfinavir
NNRTI
20
Mean
0.51
0.43
0.19
0
-0.5
0
0.5
1
1.5
Log10 viral RNA (copies/ml)
2
2.5
HIV-1 RNA copies/ml
Correlation Between
Baseline and Persistent Viremia at Week 60
Persistent Viremia in Patients on
Suppressive ART: Longitudinal Analysis
•
Abbott M97-720 Study
•
Long term observational study lopinavir/r treated patients (N=40)
•
D4T/3TC/ lopinavir/ritonavir therapy
•
Long term evaluation ≥ 7 y
Longitudinal analysis
reveals HIV-1
an additional
Late Stage
RNAthird and fourth
phase
decayTwo Phases
Decay Occurs
in of
atviral
Least
Plasma HIV-1 RNA (log10 copies/mL)
2.0
1.5
1.0
0.5
T1/2 = 63 Weeks
0.0
T1/2 = ∞
-0.5
Mixed effects model
-1.0
0
60
120
180
Week
240
300
360
102
(copies/ml)
HIV-1 RNA
Probing the mechanism of chronic viremia using
antiretroviral intensification
101
Intensification
30 day
100
NO Ongoing
Replication
Ongoing
Replication
Time
Enrollment
• Suppressed in commercial assays>1 y
• SCA ≥ 1 copy/ml
• No prior ARV resistance
NNRTI or PI Intensification Does
NOT Decrease Persistent Viremia
Dinoso et al., 2009
Raltegravir Intensification Does NOT
Decrease Persistent Viremia
PreIntensification
HIV-1 RNA
(log10 copies/ml plasma)
2
PostIntensification
Raltegravir
0.92
0.73
1.5
1
0.5
0
-0.5
-1
-30
-20
-10
0
10
20
30
40
50
60
70
Time (days)
McMahon, CID, 2010
ARV Intensification Does NOT
Decrease Persistent Viremia
• Antiretroviral intensification DOES NOT
reduce HIV-1 plasma viral RNA levels
• EFV
• ATV/r
• LPV/r
• RVR
• Selected patient population
But…
2 LTR Circles
13/45
RTG
0/24
Control
Nature Med 2010
Detecting HIV Replication in Reservoirs
• Anatomic compartmentalization is NOT well understood
Anatomic
Reduced ARV Penetration = Ongoing Replication
Charter Study Best et al., AIDS 2009
CNS
GALT
Genetically Distinct Populations
GU
Wong, Brain 2006
Detecting HIV Replication in Reservoirs
• Anatomic compartmentalization is NOT well understood
ACTG 5201
Open Label Pilot of Regimen Simplification
CNS
Swindells JAMA 2006
Wilkin, J.Inf.Dis. 2009
ENTRY
N=36
Suppressed≥ 48 weeks on combination ARV
INTERVENTION:
REGIMEN SIMPLIFIED TO r/ATZ ALONE
GALT RESULTS:
31/34 suppressed at 24 weeks
97% of all time points <50 c/ml
Resistance did not emerge in most with rebound
SCA Detected increased viremia in rebound
GU
NOT in patients with continued suppression
Similar clinical data in randomized studies of r/darunavir
monotherapy vs combination ARV (MONET), and r/Kaletra
monotherapy vs combination therapy
Characteristics of HIV During
Suppressive Therapy
• Persistent Viremia
• Quantifyable in c. 80% of patients
• Relatively stable steady state
• Third phase decline (t1/2 c.63 wk) and fourth phase (no
decline) with prolonged therapy
• Level of viremia is NOT correlated with drug regimen
• ARV therapy is potent and suppresses HIV >104-fold
• Level of viremia IS correlated with level of pretherapy
viremia
• Dynamic changes in HIV replication are reflected in level
of viremia and detectable using SCA
Quantitative Measures For Clinical
Studies of HIV Reservoirs
• HIV nucleic acid analysis
• HIV population genetics
Genetic Analysis of HIV RNA To Detect
Ongoing Replication
NO Ongoing Replication
Ongoing Replication
Divergence
Pretherapy
During therapy
Divergence
Genetic Analysis of HIV RNA To Detect
Ongoing Replication
Time (days)
Analysis of HIV Viremia After
Prolonged Suppression
• Composition of the plasma virus during
suppressive therapy
Persaud JAMA 2000
Similar Genetic Diversity and Population Structure Before and After
NO genetic
evidence
of ongoing
Initiation
of Antiretroviral
Therapyreplication
D4T/3TC/EFV
107
during ARV suppression
1000
900
800
105
700
600
104
500
103
400
300
102
200
101
100
1
0
50
100
150
200
Time on Study (days)
250
300
0
CD4 (cells/µl)
HIV-1 RNA (copies/ml)
106
1019.510
1030.723
1019.522
145242-12
1019.516
1019.57
1206.97
1115.89
1115.81
1115.810
1206.95
145241-9
145242-3
145241-10
145241-14
1115.816
1115.83
145241-13
1206.914
1115.820
1206.98
145242-1
1115.814
1030.71
1019.517
1019.52
1030.716
HIV Drug Resistance Program
NCI–Frederick
0.001 substitutions/site
Analysis of HIV Viremia After Prolonged
Suppression
Distribution of HIV diversity
Predominant
Plasma Clone (PPC)
HIV cellular DNA
HIV in plasma
HIV from resting CD4
• Repeated isolation
• Identical sequence
• NOT present in resting
CD4
• NOT major constituent of
cellular DNA
Loss of other shorter lived cells
exposed rare PPC-producing cell(s)?
Pool of cells undergoing expansion?
Bailey et al., 2006
Characteristics of HIV During
Suppressive Therapy
• HIV population genetics
• HIV populations are genetically diverse
• Do not undergo genetic bottleneck upon introduction of
ARV
• Genetic variation is markedly restricted during
suppressive therapy
• Suggest little or no active replication during therapy
Eradication Strategies
• Requirements
• Maintain suppression of active HIV-1 replication
• Continue ARV during eradication
• Dual approach
•Critical
Target cellsTest
with low
HIV-1 production
oflevel
Eradication:
• Ensure activation of cells with “latent” HIV
Interrupt Antiretrovirals
infection
• Permanent silencing for durable effect
Eradication Strategies
• Detecting HIV during suppressive therapy and
eradication strategies
• Sensitive detection systems
• Single copy nucleic acid detection
•RNA
•DNA
• IUPM
• Genetic analyses
• Robust performance characteristics
•Poisson limitations
• Patient selection and characterization is essential
• Useful assays are essential to ensure patient
safety
Acknowledgments
HIV Drug Resistance
Program
•
•
•
•
•
•
•
•
•
•
•
•
•
S. Hughes
J. Coffin
M. Kearney
A. Wiegand
V. Boltz
W. Shao
J. Spindler
H. Mens
S. Yu
N. Urban
F. Cossarini
C. Poethke
Karoll Cortez
University of Pittsburgh
• J. Mellors
• D. McMahon
• J. Jones
NIAID/CCMD Clinic
•
•
•
•
•
•
•
•
•
•
•
•
•
•
H. C. Lane
H. Masur
R. Davey
M. Polis
J. Kovacs
J. Mican
I. Sereti
S. Migueles
A. O’Shea
C. Rehm
R. Dewar
S. Mitchell
J. Metcalf
Clinical Fellows
Patient Volunteers
Tufts University
• John Coffin
Johns Hopkins
University
•
•
•
J. Dinoso
S. Gange
R. Silicano
Karolinska
Institute
• S. Palmer
Abbott Lab.
• M. King
• S. Brun
• D. Kempf
• G. Hanna
Eradication Strategies
• Stimulate HIV expression from latently infected cells
• HDAC and other approaches to remodel chromatin
• Specific HIV induction
• Immune modulators
• Target infected cells with low level replication
• Inhibit cellular activation
• Direct cytotoxic therapy
• Gene therapy approaches
• Transplantation approaches
• Replacement of bone marrow with HIV resistant donor
• Heller et al., 2009
• NOT widely applicable
•
•
ARV discontinuation
Clinical success will require surveillance
Nature of reservoir requires distinct approaches to
eradication
Status of HIV Infected
Cell During Therapy
Constitutive HIV Replication
“LATENT”
Chromatin
Remodeling
HIV mRNA
HIV mRNA
+1
U3
Transcription
Factors
Inducible HIV Replication
+1
R
U5
Target HIV Directly
antigen stimulation
Transcription
U3
R
Factors
U5
Target HIV Indirectly
Activate Chromatin
Remodeling
Similar Genetic Diversity and Population Structure Before and After
NO genetic
evidence
of ongoing
Initiation
of Antiretroviral
Therapyreplication
D4T/3TC/EFV
107
during ARV suppression
1000
900
800
105
700
600
104
500
103
400
300
102
200
101
100
1
0
50
100
150
200
Time on Study (days)
250
300
0
CD4 (cells/µl)
HIV-1 RNA (copies/ml)
106
1019.510
1030.723
1019.522
145242-12
1019.516
1019.57
1206.97
1115.89
1115.81
1115.810
1206.95
145241-9
145242-3
145241-10
145241-14
1115.816
1115.83
145241-13
1206.914
1115.820
1206.98
145242-1
1115.814
1030.71
1019.517
1019.52
1030.716
HIV Drug Resistance Program
NCI–Frederick
0.001 substitutions/site
HIV Reservoirs:
Distinct Subsets Diverse Activation
Signalling
• Central Memory
• Transitional Memory
3
T
HIV Eradication
Anti-Latency Strategies
+1
R
AP-1
ATF/CREB
U5
AP-3
NFAT
HIV Eradication
Anti-Latency Strategies
TAR
SP
AP-3
NFAT
NRE
U3
C/EBP
NF-κB
SP
+1
TATA
AP-3
AP-1
ATF/CREB NFAT
R
U5
HIV Eradication
Anti-Latency Strategies
TAR
SP
AP-3
NFAT
NRE
U3
C/EBP
NF-κB
SP
+1
TATA
AP-3
AP-1
ATF/CREB NFAT
R
TBP
associated factors
U5
HIV Eradication
Anti-Latency Strategies
TAR
SP
AP-3
NFAT
C/EBP
NF-κB
SP
+1
TATA
NRE
U3
AP-3
AP-1
ATF/CREB NFAT
R
SP/KLF
Zn++ Finger binding
U5
HIV-1 Suppression by Transplant
ARV
Hutter et al., NEJM, 2009
ARV
106
HIV RNA
copies/ml104
102
-206
-4
+108
Chemotherapy Conditioning/Transplant
+332
+548
Conditioning/Transplant
Multiphase HIV decay to therapy
Engraftment with ΔCCR5
No viremia off ART but leukemic failure
Second transplant controlled leukemia
Elimination of reservoir by replacement AND…
Graft vs HIV infected cell effect?
All latent infected cells undergo activation OR
All infected cells are detectable by graft
HIV Eradication Strategies
• Neoplastic diseases therapy as paradigm
• Successful especially when tumor burden
is substantial
• Relevance to low frequency targets like
HIV infected cells depends on specificity
Nature of reservoir requires distinct approaches to
eradication
Status of HIV Infected Cell During Therapy
Constitutive HIV Replication
• Anti-CD45 Ro
• Zeta chain therapy
HIV mRNA
+1
U3
Transcription
Factors
Targeting Low Level
HIV Production
• Pseudomonas exotoxin targeting Env
R
U5
Target HIV Directly
Nature of reservoir requires distinct approaches to
eradication
Status of HIV Infected Cell During Therapy
Inducible HIV Replication
“LATENT”
Chromatin
Remodeling
HIV mRNA
+1
antigen stimulation
Transcription
U3
R
Factors
U5
Excellent models in vitro
• Cell lines
• Lymphocytes ex vivo
Numerous potential strategies
• Integration site selection
• Chromosome modeling
•Valproate
• Transcriptional approaches
• Post transcriptional approaches
Active agents with potential
Target HIV Indirectly
Activate Chromatin
Remodeling
• Disrupt nucleic acid sites required
for activation
• Disrupt nucleic acidactivator interactions
• Modulate activation and expression of
activators
Nature of reservoir requires distinct approaches to
eradication, unless we just target everything
Status of HIV Infected Cell During Therapy
“LATENT”
Chromatin
Remodeling
HIV mRNA
+1
antigen stimulation
Transcription
U3
R
Factors
U5