hiv drug resistance program nci–frederick targeting ‘residual hiv’ in its reservoirs: where we...

HIV Drug Resistance ProgramNCI–Frederick

Targeting ‘Residual HIV’ In Its Targeting ‘Residual HIV’ In Its Reservoirs: Where We Are And Reservoirs: Where We Are And

Where Do Where Do WeWe Go? Go?

HIV Reservoirs WorkshopHIV Reservoirs Workshop Vienna, Austria Vienna, Austria

July 17,2010 July 17,2010 Frank Maldarelli, M.D., Ph.D.Frank Maldarelli, M.D., Ph.D.

Targeting ‘Residual HIV’ In Its Targeting ‘Residual HIV’ In Its Reservoirs: Where We Are And Reservoirs: Where We Are And

Where Do Where Do WeWe Go? Go?

HIV Reservoirs WorkshopHIV Reservoirs Workshop Vienna, Austria Vienna, Austria

July 17,2010 July 17,2010 Frank Maldarelli, M.D., Ph.D.Frank Maldarelli, M.D., Ph.D.

Time (weeks)

HIV-1RNA

HIV Response to Antiretroviral Therapy

Detection limit

101

102

103

104

105

0 4 8 16

ARVARV

Decay Kinetics of Viral Infected Cells

HIV-1 Infected Cells

Half life of infected cells (days)1 Longer14

Detection limit

104

105

106

107

108

Activated Lymphocyte

Longer lived cells

Macrophage?

Identifying the source of HIV viremia during suppressive antiretroviral therapy is essential to

eradication

Identifying the source of HIV viremia during suppressive antiretroviral therapy is essential to

eradication

Stable reservoirs

NEW STRATEGIES NEW STRATEGIES

NEEDEDNEEDED

HIV production from HIV production from reservoirs is NOT reservoirs is NOT

blocked by ARV therapyblocked by ARV therapy

IMPROVED ARV NEEDEDIMPROVED ARV NEEDED

Active replication cycles

Infected cell Uninfected cellInfected cell Uninfected cell

HIV production from HIV production from active replication is active replication is

blocked by ARV therapyblocked by ARV therapy

X

Quantitative Measures For Clinical Studies of HIV Reservoirs


• HIV nucleic acid analysis

• HIV population genetics

Single Copy Quantitation of HIV-1 Single Copy Quantitation of HIV-1 ViremiaViremia

Single Copy Quantitation of HIV-1 Single Copy Quantitation of HIV-1 ViremiaViremia

• 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

Per

cen

t m

ain

tain

ing

vi

rolo

gic

res

po

nse

nelfinavir

lopinavir/ritonavir

21% Failure

44% Failure

Week

p<0.001, Cox proportional hazards model

Superior Efficacy of Lopinavir/ritonavir over Nelfinavir

Abbott 98-863 Study

Superior Efficacy of Lopinavir/ritonavir over Nelfinavir

Abbott 98-863 Study

Does a difference in antiviral potency impact viremia on therapy?

Walmsley, S. N. Engl. J. Med., 2002Walmsley, S. N. Engl. J. Med., 2002

Selected 130 patients (67 NFV, 63 LPV/r)Remained <50 copies/ml following wk 24

0

20

40

60

80

100

-0.5 0 0.5 1 1.5 2 2.5

Log10 viral RNA (copies/ml)

Dis

trib

uti

on

Ran

k (

Per

ce

nti

le)

nelfinavir 0.48 0.43 NNRTI 0.35 0.19

Viremia on Therapy is Independent of RegimenViremia on Therapy is Independent of Regimen

lopinavir/ritonavir 0.53 0.51 Median MeanMedian Mean

Correlation BetweenBaseline and Persistent Viremia at Week 60

HIV

-1 R

NA

co

pie

s/m

lH

IV-1

RN

A c

op

ies/

ml

Persistent Viremia in Patients on Suppressive ART: Longitudinal Analysis

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

Late Stage HIV-1 RNADecay Occurs in at Least Two Phases

Late Stage HIV-1 RNADecay Occurs in at Least Two Phases

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

0 60 120 180 240 300 360

Week

Pla

sma

HIV

-1 R

NA

(lo

g 10

co

pie

s/m

L)

Longitudinal analysis reveals an additional third and fourth phase of viral decay

Longitudinal analysis reveals an additional third and fourth phase of viral decay

T1/2 = 63 Weeks T1/2 = ∞

Mixed effects modelMixed effects model

101022

101011

101000

HIV

-1 R

NA

(co

pie

s/m

l)

TimeTime

Probing the mechanism of chronic viremia using antiretroviral intensification

Probing the mechanism of chronic viremia using antiretroviral intensification

NO Ongoing Replication

Ongoing Replication

30 day

IntensificationIntensification

Enrollment• Suppressed in commercial assays>1 y• SCA ≥ 1 copy/ml • No prior ARV resistance

NNRTI or PI Intensification Does NNRTI or PI Intensification Does NOT Decrease Persistent ViremiaNOT Decrease Persistent ViremiaNNRTI or PI Intensification Does NNRTI or PI Intensification Does

NOT Decrease Persistent ViremiaNOT Decrease Persistent Viremia

Dinoso et al., 2009

Raltegravir Intensification Does NOT Raltegravir Intensification Does NOT Decrease Persistent ViremiaDecrease Persistent Viremia

-1

-0.5

0

0.5

1

1.5

2

-30 -20 -10 0 10 20 30 40 50 60 70

Raltegravir

HIV

-1 R

NA

(l

og

10 c

op

ies

/ml p

las

ma

)

Time (days)

Pre-Intensification

Post-Intensification

0.920.92 0.730.73

McMahon, CID, 2010

• Antiretroviral intensification DOES NOT reduce HIV-1 plasma viral RNA levels

• EFV• ATV/r• LPV/r• RVR

• Selected patient population

ARV Intensification Does NOT ARV Intensification Does NOT Decrease Persistent ViremiaDecrease Persistent Viremia

ARV Intensification Does NOT ARV Intensification Does NOT Decrease Persistent ViremiaDecrease Persistent Viremia

But…But…

Nature Med 2010

2 LTR Circles

13/45 RTG

0/24 Control

Detecting HIV Replication in ReservoirsDetecting HIV Replication in Reservoirs

• Anatomic compartmentalization is NOT well understood

CNS

GALT

GU

Anatomic

Reduced ARV Penetration = Ongoing Replication Charter Study Best et al., AIDS 2009

Wong, Brain 2006

Genetically Distinct Populations

Detecting HIV Replication in ReservoirsDetecting HIV Replication in Reservoirs

• Anatomic compartmentalization is NOT well understood

CNS

GALT

GU

ACTG 5201 ACTG 5201 Open Label Pilot of Regimen SimplificationOpen Label Pilot of Regimen Simplification

Swindells JAMA 2006 Wilkin, J.Inf.Dis. 2009ENTRYENTRY N=36N=36 Suppressed≥ 48 weeks on combination ARVSuppressed≥ 48 weeks on combination ARVINTERVENTION: INTERVENTION: REGIMEN SIMPLIFIED TO r/ATZ ALONEREGIMEN SIMPLIFIED TO r/ATZ ALONERESULTS:RESULTS: 31/34 suppressed at 24 weeks31/34 suppressed at 24 weeks 97% of all time points <50 c/ml97% of all time points <50 c/ml Resistance did not emerge in most with reboundResistance did not emerge in most with rebound SCA Detected increased viremia in reboundSCA Detected increased viremia in rebound NOT in patients with continued suppressionNOT in patients with continued suppression

Similar clinical data in randomized studies of r/darunavir Similar clinical data in randomized studies of r/darunavir monotherapy vs combination ARV (MONET), and r/Kaletra monotherapy vs combination ARV (MONET), and r/Kaletra monotherapy vs combination therapymonotherapy vs combination therapy

Characteristics of HIV During Characteristics of HIV During Suppressive TherapySuppressive 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



• HIV nucleic acid analysis

• HIV population genetics

Genetic Analysis of HIV RNA To Detect Ongoing Replication


Pretherapy

During therapy

Divergence

NO Ongoing ReplicationNO Ongoing Replication Ongoing Replication

Divergence



Time (days)

Analysis of HIV Viremia After Prolonged Suppression


• Composition of the plasma virus during suppressive therapy

Persaud JAMA 2000


101

HIV

-1 R

NA

(co

pie

s/m

l)

200

300

400

500

600

700

800

900

1000

CD

4 (c

ells

/µl)

D4T/3TC/EFV

1 0

100

1019.5101030.7231019.522

145242-12

1019.5161019.57

1206.971115.891115.81

1115.8101206.95145241-9145242-3

145241-10145241-14

1115.8161115.83145241-131206.914

1115.8201206.98145242-1

1115.8141030.71

1019.5171019.52

1030.716

0.001 substitutions/site

102

103

104

105

107

106

Time on Study (days) 0 50 100 150 200 250 300

Similar Genetic Diversity and Population Structure Before and After Similar Genetic Diversity and Population Structure Before and After Initiation of Antiretroviral TherapyInitiation of Antiretroviral TherapyNO genetic evidence of ongoing replication during ARV suppression


Bailey et al., 2006

Predominant Plasma Clone (PPC)HIV cellular DNA

HIV in plasmaHIV from resting CD4

Distribution of HIV diversity

• Repeated isolationRepeated isolation• Identical sequenceIdentical sequence• NOT present in restingNOT present in resting CD4CD4• NOT major constituent ofNOT major constituent of cellular DNAcellular DNA

Loss of other shorter lived cells exposed rare PPC-producing cell(s)?

Pool of cells undergoing expansion?



• 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

• Requirements• Maintain suppression of active HIV-1 replication• Continue ARV during eradication

• Dual approach• Target cells with low level HIV-1 production• Ensure activation of cells with “latent” HIV

infection• Permanent silencing for durable effect

Eradication StrategiesEradication Strategies

Critical Test of Eradication: Interrupt Antiretrovirals

• 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


AcknowledgmentsAcknowledgmentsNIAID/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

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

Tufts University • John Coffin

Karolinska Institute

• S. PalmerS. Palmer

Abbott Lab.• M. King• S. Brun• D. Kempf• G. Hanna

Johns Hopkins University

• J. Dinoso• S. Gange• R. Silicano

Patient VolunteersPatient Volunteers

• 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


antigen stimulation

Status of HIV Infected Cell During TherapyStatus of HIV Infected Cell During Therapy

Constitutive HIV ReplicationConstitutive HIV Replication Inducible HIV ReplicationInducible HIV Replication

U3 R U5

“LATENT”

U3 R U5

Chromatin Remodeling

Target HIV Directly Target HIV IndirectlyActivate Chromatin

Remodeling

Nature of reservoir requires distinct approaches to eradication


+1

HIV mRNA

+1

HIV mRNA

Transcription

Factors Transcription

Factors


101

HIV

-1 R

NA

(co

pie

s/m

l)

200

300

400

500

600

700

800

900

1000

CD

4 (c

ells

/µl)

D4T/3TC/EFV

1 0

100

1019.5101030.7231019.522

145242-12

1019.5161019.57

1206.971115.891115.81

1115.8101206.95145241-9145242-3

145241-10145241-14

1115.8161115.83145241-131206.914

1115.8201206.98145242-1

1115.8141030.71

1019.5171019.52

1030.716

0.001 substitutions/site

102

103

104

105

107

106

Time on Study (days) 0 50 100 150 200 250 300

Similar Genetic Diversity and Population Structure Before and After Similar Genetic Diversity and Population Structure Before and After Initiation of Antiretroviral TherapyInitiation of Antiretroviral TherapyNO genetic evidence of ongoing replication during ARV suppression

HIV Reservoirs:Distinct Subsets Diverse Activation

Signalling

HIV Reservoirs:Distinct Subsets Diverse Activation

Signalling

• Central Memory

• Transitional Memory

HIV Eradication Anti-Latency Strategies


U5

R

+1

AP-1ATF/CREB

AP-3NFAT

AP-3NFAT



R U5

AP-1ATF/CREB

AP-3NFAT

NRE

AP-3NFAT

C/EBP NF-κB SP TATASP

U3

+1TAR



R U5

AP-1ATF/CREB

AP-3NFAT

NRE

AP-3NFAT


U3

+1TAR

TBP associated factors



R U5

AP-1ATF/CREB

AP-3NFAT

NRE

AP-3NFAT


U3

+1TAR

SP/KLF

Zn++ Finger binding

HIV-1 Suppression by TransplantHIV-1 Suppression by TransplantHutter et al., NEJM, 2009

102

104

106

HIV RNAcopies/ml

-206 -4 +108 +548+332

Chemotherapy Conditioning/Transplant

ARV ARV

Conditioning/Transplant

Engraftment with ΔCCR5No viremia off ART but leukemic failureSecond transplant controlled leukemia

Multiphase HIV decay to therapy

Elimination of reservoir by replacement AND…Graft vs HIV infected cell effect?

All latent infected cells undergo activation ORAll infected cells are detectable by graft

HIV Eradication StrategiesHIV 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


Constitutive HIV ReplicationConstitutive HIV Replication

U3 R U5

Target HIV Directly



+1

HIV mRNA

Transcription

Factors

Targeting Low Level HIV Production

• Anti-CD45 Ro• Zeta chain therapy

• Pseudomonas exotoxin targeting Env

antigen stimulation


Inducible HIV ReplicationInducible HIV Replication

“LATENT”

U3 R U5


Target HIV IndirectlyActivate Chromatin

Remodeling



+1

HIV mRNA

Transcription

Factors

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• Disrupt nucleic acid sites required

for activation• Disrupt nucleic acid-

activator interactions• Modulate activation and expression of activators

antigen stimulation


“LATENT”

U3 R U5


Nature of reservoir requires distinct approaches to eradication, unless we just target everything

Nature of reservoir requires distinct approaches to eradication, unless we just target everything

+1

HIV mRNA

Transcription

Factors

hiv drug resistance program nci–frederick targeting ‘residual hiv’ in its reservoirs: where we...

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