Towards an ART-less Future

The Challenge of a Cure for HIV Infection

David M. Margolis, MDProfessor of Medicine, Microbiology & Immunology, Epidemiology

Lohse, N. et. al. Ann Intern Med 2007;146:87-95Lohse, N. et. al. Ann Intern Med 2007;146:87-95

Antiretroviral therapy: >3 million years of life savedAntiretroviral therapy: >3 million years of life saved

Predicted Survival if HIV+ at age 25 yearsPredicted Survival if HIV+ at age 25 yearsPredicted Survival if HIV+ at age 25 yearsPredicted Survival if HIV+ at age 25 years

More than two decades ofMore than two decades ofpandemic HIV:pandemic HIV:

what is to be done in the coming decades?what is to be done in the coming decades?

• Prevent disease in the infected

• Prevent infection

• Eradicate infection

Prevent Disease in the InfectedChallenges of Long-term Antiretroviral Therapy

• Drug Resistance

• Cost

• Adherence

• Long-term Toxicities

For every patient that starts therapy,3 to 4 new infections occur

For every patient that starts therapy,3 to 4 new infections occur

Prevent infection

Education and behavior modificationCondoms, and other barrier methodsTreatment/prevention of drug/alcohol abuseClean syringes (i.e. needle exchange programs)Interruption of mother-to-child transmissionCircumcisionHIV/STI TestingAntiretroviral treatment as preventionPre-exposure prophylaxis (PrEP)Topical microbicidesVaccination

Prevent infection

Education and behavior modificationCondoms, and other barrier methodsTreatment/prevention of drug/alcohol abuseClean syringes (i.e. needle exchange programs)Interruption of mother-to-child transmissionCircumcisionHIV/STI TestingAntiretroviral treatment as preventionPre-exposure prophylaxis (PrEP)Topical microbicidesVaccination

Pierson et al 2001

Eradicate HIV InfectionToday: Persistent HIV Infection despite ART

Maldarelli et al. PLoS Path 2007

100 copies

10 copies

1 copy

Low-level viremiaDornadula JAMA 1999Palmer et al. J Clin Micro 2003: Single-copy assay

• detected in ca. 75% ART-suppressed patients • Level correlates with peak viremia

• 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

Pre-RALPre-RAL

0.04 l0.04 l0.140.14

0.040.04

2

1

0

-1

P=0.69P=0.69 P=0.38P=0.38

HIV

-1 R

NA

(lo

g 10 c

opi

es/m

L)H

IV-1

RN

A (

log 1

0 c

opi

es/m

L)

No Decrease in Residual Viremia during Raltegravir Intensification in Patients on Standard ART Jones, et al. PNAS 2009

RALRAL Post-RALPost-RAL

Unaffected by intensification with PI, NNRTI, RAL, Enf

• 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 DNAIntegrated HIV DNATotal 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)

Transient Increase in Episomal Viral cDNA following Raltegravir Intensification of a Stable HAART RegimenM Buzon, J Llibre, J Gatell, P Domingo, R Paredes, S Palmer, M Sharkey, M Stevenson, B Clotet and Javier Martinez-Picado

CROI 2009; Abstr. 423aCROI 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

Pierson et al 2001

Rare, persistent proviral genomes

Persistent HIV Infection despite ART

Maintenance

Activation

Targeting latency in resting CD4+ T cells

HIV-infectedResting

CD4+ T cellsK in K out

ART

K out

Establishment

Derepression

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

IBIB

HMBA

Pros

trat

in

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

• Host miRNA: HIV RNA translational effect, HIV LTR heterochromatin effect

• Resting cell deficient in RNA export factor: PTB

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.

deacetylated

HIVlives

withinchromatin

“Closed” NucleosomeGene Expression Repressed

“Open” HistonesGene Expression Active

acetylated

Nuc-1Nuc-2

Nuc-0

LTR

“Opening” of Chromatin is Required forHIV Expression

Nuc-1Nuc-2

Nuc-0

Van Lint, Emiliani, Ott, Verdin 1996Sheridan, Mayall, Verdin, Jones 1997

El Kharroubi, Piras, Zensen, Martin 1998

Histoneacetyl-

transferase

NF-B, Sp1

Nuc-1Nuc-2

Nuc-0

LTR

Margolis et al., J Virol 1994Romerio et al., J Virol 1997Coull et al., J Virol 2000He & Margolis, MCB 2002Ylisastigui et al. JID 2002

Histonedeacetylases

can shut downHIV expression

LSF LSF

YY1

HDAC1

Nuc-1

deacetylated

HIVlives

withinchromatin

“Closed” NucleosomeGene Expression Repressed

“Open” HistonesGene Expression Active

acetylated

Nuc-1

Redundancy in HDAC1 action at the LTR

NF-B, Sp1

Nuc-2Nuc-0

HDAC1 recruitment by YY1/LSFRomerio et al., J Virol 1997

Coull et al., J Virol 2000He & Margolis, MCB 2002

Coull et al. J Virol 2002Ylisastigui et al. JID 2002

LTR

HDAC1 recruitmentby p50 of NF-kBWilliams et al. 2006

HDAC1 recruitmentby Sp1/c-MycJiang et al. 2007

HDAC1 recruitmentby CBF-1

Tyagi & Karn 2007

HDAC1 recruitmentby Ap4

Imai & Okamoto 2006

Expected decayt1/2=44 mo.

420

10

100

6Months on protocol therapy

IUPB

Resting

CD4+

T cells

Patient 2

IUPB

Resting

CD4+

T cells

420 610

100

Patient 1

100

1000

420 6

Patient 3

Months on protocol therapy420

10

100

6

Patient 4

49250

57136

35

81

< 9

31

Effect of VPA and Enfuvirtide on Resting CD4+ cell infection (per billion)

Lehrman et al. Lancet 2005

Stability of the Latent Reservoir for HIV-1 in Patients Receiving Valproic Acid. Siliciano et al., J Infectious Diseases 2007;195:833-836

Prolonged valproic acid treatment does not reduce the size of latent HIV reservoir. Sagot-Lerolle, N. et al. AIDS 2008; 22:1125-1129

* 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

Infected resting CD4+ T cells per billion1-copy assay 

Patient ART IUPB

ART and VPA IUPB

>50% depletion IUPB on VPA range median

1 2 3 4

187 52 70

183

90 22 22 3

Yes Yes Yes Yes

<1 - 1 <1 - 3 <1 - 5 n.d.

<1 1 2

n.d. 5 6 7 8 9

10 11

112 54

531 17951 4890 696 57

120 75

625 9438 3300 1253

83

No No No

No* No* No No

n.d. <1 - 1 1 - 7

8 - 77 24 - 87

<1¤ <1 Š 1¤

--- <1 3

25 37 --- ---

Valproic acid without intensified ART has a limited effect on resting CD4 T cell infection. Archin et al. AIDS 2008

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 inpatient’s resting CD4+ T cells

Class I Class II Class IV

Keedy J Virol 2009

N C N C Rest Act

HDAC8

N C N C Rest Act

HDAC1

N C N C Rest Act

HDAC2

N C N C Rest Act

HDAC3

Lamin B1:

GAPDH:

N C N C Rest Act

HDAC11

Lamin B1:

GAPDH:

Class I Class IV

N C N C Rest Act

HDAC4

N C N C Rest Act

HDAC5

N C N C Rest Act

HDAC6

Lamin B1:

GAPDH:

N C N C Rest Act

HDAC7

N C N C Rest Act

HDAC9

N C N C Rest Act

HDAC10

Class II

Nuclear localization of HDACs 1, 2 & 3 and 4, 6 & 7in patient’s resting CD4+ T cells

Keedy J Virol 2009

α-A

cH3

α-H

DAC1

α-H

DAC2

α-H

DAC3

IgG

10% Input

IP:

TSA: - + - + - + - +

IP: 10% Input IgG α-AcH4 α-HDAC4

IP: 10% Input IgG α-AcH4 α-HDAC6

IP: 10% Input IgG α-AcH4 α-HDAC7

HDACs 1, 2 & 3found at

the HIV LTR

HDACs4, 6 & 7

notfound

Keedy J Virol 2009

Antibodies Used:

CD4 purificationCD8, CD14, CD16CD19, CD56Glycophorin A

Resting cell cocktailCD41, CD25, HLA-DR

Mix cells withAntibody-magnetic bead

cocktail

Magnet Magnet

200-1000millionresting

CD4+ cells

4 billionlymphocytes

fromaviremic patient

on HAART

Measuring resting CD4+ T cell infection (“latency”)Resting CD4+ Cell Viral Outgrowth Assay:

Purified Resting cells

Incubated with IntegraseInhibitor & RT Inhibitor

Viral Induction

Outgrowth assay

PHA/IL2/allo

IL2

Test drugTest drug

Single donor, R5-high,CD8-depleted PBMCtargets twice a week;

p24 day 17, confirm day 19

2.5 million cells/well

2.5 million cells/well

0.5 million cells/well

0.1 million cells/well

Assayvariance:

0.3 log

Range:25 cells/millionto 9 cells/billion

Infe

cte

d u

nits

pe

r b

illio

n

rest

ing

CD

4+

T c

ells

PHA

10

100

1000

10000

Class II

inhibitor

Infe

cte

d u

nits

pe

r b

illio

n

rest

ing

CD

4+

T c

ells

PHA Class Iinhibitor

10

100

1000

10000

Infe

cte

d u

nits

pe

r b

illio

n

rest

ing

CD

4+

T c

ells

PHA VPAglobal HDAC

inhibitor

10

100

1000

10000

100000

Infe

cte

d u

nits

pe

r b

illio

n

rest

ing

CD

4+

T c

ells

PHA

10

100

1000

Class II

inhibitor

Limit ofdetection

Archin AIDS 2009Archin AIDS 2009

Infe

cted

un

its

per

bil

lio

n

rest

ing

CD

4+ T

cel

ls

PHA

10

100

1000

HDAC1, 2, 3

inhibitor

Archin et al AIDS 2009Archin et al AIDS 2009

Class Iinhibitor

HDAC 1, 2inhibitor

HDAC1, 2, 3

inhibitor

T Cell model system

Patient’s cell assay

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 2009Contreras 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 Modeling ART in HIV infected BLT micemice

P Denton, JV Garcia-Martinez

Prevent Infection“Test and Treat”Prevent Infection“Test and Treat”

Start ART

Target residual viremia(immunotherapy?)

Anti-latencytherapy

…..eradicationor remission

Future ChallengesFuture Challenges

What do we need to attackpersistent HIV infection?

Persistence

•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

Nancy Archin Kara KeedyManzoor Cheema Daniel ParkerRobbie Sackmann Kriston BartonShailesh Choudhary

• 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

Daria HazudaRon Bosch

Anne Weigand, John CoffinPaul Denton, Victor Garcia

siRNAs targeting HDAC2 or 3 Activate HIV Gene Expression

***

Keedy J Virol 2009

siRNAs targeting HDAC2Induce HIV Outgrowth

Keedy preliminary data

Archin 2009