The search for a cure: A drug discovery perspective on HIV eradication

“A game of hide and sleep”

Daria Hazuda, Merck and Co

Why can’t we cure HIV with ARV DrugsWhere is the virus and how is it maintained in the

face of “suppressive” therapy?

Defining the problem…..

The Reservoir of Latently Infected Cells Decays Slowly and is replenished?

Siliciano et al., Nat Med (2003) 9:727

Decay of Latent Reservoir in Patients with full HAART suppression for 3-7 years

• HIV Integrates into cells with extremely long biological half-life

• Integrated HIV persists in patients on HAART

• Latent reservoir makes HIV infection incurable

• Potential for clonal expansion of latently infected cellsand proliferation

Chomont et al, Nature Medicine, 2010

Persistent low level viremia can be detected in nearly all patients on HAART

Plasma HIV RNA (log)

Perc

en

tile

2.5

Nelfinavir

0

20

40

60

80

100

-0.5 0 0.5 1 1.5 2

Lopinavir/Ritonavir

Maldarelli, Palmer, et al. PLoS Pathogens 2007

Median 3.1 copies RNA/mL at week 60

T cell activation declines, but remains abnormal even after many years of HAART

Hunt PW, et al. J Infect Dis. 2003;187:1534-1543 and unpublished

% C

D38

+H

LA

DR

+C

D8+

T C

ells

0

20

40

60

80

HIVNegative(n=82)

Non-Controller

(n=65)

HAART(n=132)

P < 0.001

P < 0.001

The size of the HIV reservoir (defined by RNA/DNA ratio) is associated with frequency of activated

CD4+ T cells in rectal tissues

Hunt, Yukl and Wong

Multiple studies show RAL intensification does not impact residual plasma viremia

Massanella et al., CROI 2011

…but intensification may impact immune activation?

2

Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy.Yukl, Steven; Shergill, Amandeep; McQuaid, Kenneth; Gianella, Sara; Lampiris, Harry; Hare, C; Pandori, Mark; Sinclair, Elizabeth; Gunthard, Huldrych; Fischer, Marek; Wong, Joseph; Havlir, Diane

AIDS. 24(16):2451-2460, October 23, 2010.DOI: 10.1097/QAD.0b013e32833ef7bb

Fig. 2 . Change in cell-associated unspliced HIV RNA (a, b) and HIV DNA (c) per 106 CD4+ T cells. HIV copy numbers were measured by real-time PCR, normalized for the total cell input into the PCR (by [mu]g RNA or DNA), and then normalized to the percentage of all cells that were CD4+ T cells (by flow cytometry). (b) Shows the HIV RNA values in the ileum for each participant at weeks 0 and 12. In (a) and (c), column heights indicate the average of the changes (week 12-week 0) in HIV copy number per 106 CD4+ T cells, as measured from peripheral blood mononuclear cells or total gut cells (obtained by collagenase digestion of endoscopic biopsies) from each of the four gut sites. Error bars indicate the standard error of measurement (SEM).

Is tissue the issue?

Cervicovaginal Fluid Exposure mean percent of blood plasma

600%

500%

400%

300%

200%

100%

75%

50%

25%

0%

TDF (110%)

3TC (400%)

FTC (375%)

ZDV (235%)

ddI (21%)

ABC (8%)

d4T (5%)

DLV (20%)

NVP (80%)

EFV (0.4%)

N(t)RTIs NNRTI PI Entry Inhibitors

MVC (410%)

IDV (200%)

APV (50%)

RTV (26%)

ATV (18%)

LPV (8%)SQV (ND)

Integrase Inhibitors

AUC GT = AUC BP

RAL (93%)

Dumond,J et al. AIDS 2007

Why can’t we cure HIV with ARV DrugsWhere is the virus and how is it maintained in the

face of “suppressive” therapy?

Residual replication (Sanctuaries; drug penetration)

Persistent HIV expression

Immune dysfunction

Latently infected reservoir

Homeostatic Proliferation

Inflammation

These are not mutually exclusive mechanisms; will multiple approaches be required?

A game of hide and sleep?

“Shock and Kill” ApproachRationale and Goal

Hypothesis– Reactivation of HIV-1 within latent reservoirs in the

presence of HAART will lead to elimination of latent reservoirs through a combination of cytopathic viral and immune mechanisms

Goal– Use small molecule(s) to reactivate latent HIV-1 genomes,

purge the reservoir and elicit a “sustained virologic response” in the absence of continued antiretroviral therapy

Induction/eradication

“Open” HistonesAcetylated Histone tails

Reduced Higher Order StructureAccess to Transcription Factors

Transcription Enabled

“Closed”NucleosomeHypo-Acetylated Histone tails

Stable, Compact ChromatinLess accessible to Transcription Factors

Transcription Repressed

deacetylated

acetylated

HIV lives within chromatinLatency restriction at transcription initiation

Proposed mechanisms to affect latent proviral HIV-1 expression

Adapted from Richman et al, 2009

Me

MTInhibitor

MT

MeMT

HIV LatencyCell Culture Models

Integrated LTR-reporter constructs– Advantages: amenable to screening– Disadvantages: highly reductionist

Chronically infected, inducible cell lines– Advantages: complete integrated HIV genome– Disadvantages: clonal, a single integration site

Retroviral vectors – Advantages: Mixed population– Disadvantages: heterochromatin; mixed population

Ex-vivo infected primary resting CD4+ T cells Resting CD4+ T cells from HIV+ aviremic patients

throughput

biological relevance

Research & Discovery Process

FDA Review 1

5,000 – 10,000 Compounds

250 Compounds

5 Compounds

Drug Discovery

ClinicalTrials

Post-Marketing

Preclinical

2

Years

Phase IIIn=1000-5000

Phase IIn=100-500

Phase In=20-100

1DiMasi JA, Hansen RW, Grabowski HG Journal of Health Economics 22 (2003): 151-185

6

1.5

5

Incr

easi

ng b

iolo

gica

l com

plex

ity

Screening Millions of Compounds

Merck High Thoughput Screen Assay for Activators of Latent HIV-1 Gene Expression

24 hours

Add Compounds

Hela P4/R5 cells

24 hours

b-Galactosidase Assay

E.coli lacZHIV LTR polyA

b-galactosidaseUninfected cell

E.coli lacZHIV LTR polyA

b- galactosidase

Uninfected cell + compound

Activation of HIV-1 Gene Expression Correlates with HDACi Potency

~ 1.5 million compounds (MRL Library)

~ Confirmed 104 compounds (not known HDACIs)

~ 92 compounds that did not activate T-cell

~ 83 compounds with potential novel mechanism

LTR-bGal screen

NFAT Jurkat cell assay

HDAC activity assay (novel HDACis)

ToxicityChemical attractivenessFurther chacterization eg ACH-2, J1.1, primary cells, ex vivo

Non-mechanism based screening can identify novel HIV-1 activators

Characterizing known vs novel activators

^ TW Chun; D Margolis; * Planelles et al, 2008; Yang et al, 2010; Tyagi et al, 2010; In-house Merck data** Archin et al, 2009; Kutsch et al, 2002; Reuse et al, 2009; Williams et al, 2004; Burnett et al, 2010 # In-house Merck data

+/-

Are these meaningful differences or a reflection of the spectrum of relevant biology?

Patient cells Ex Vivo Jurkat line Hela Toxicity

HDACi VPA

HDACiSAHA

pTEFbHBMA

NFkBProstratin

NFkBTNF

Novel ????

Induction and eradication

Will induction lead to eradication?

Following removal of induction stimuli, cells can return to resting state in vitro

The inability to eradicate HIV-1 infection in the face of suppressive ARV therapy is a consequence a dynamic, latently infected reservoir and an anergic immune response.

Induction and eradication

Will induction lead to eradication?

Following removal of induction stimuli, cells can return to resting state in vitro

The inability to eradicate HIV-1 infection in the face of suppressive ARV therapy is a consequence a dynamic, latently infected reservoir, persistent viremia and an anergic immune response.

Eradication will require multiple approaches in combination

Can you “reset” the immune system without therapy intensification and shutting off persistent antigen production?

InflammationActivationProliferation

Latent reservoir Residual Replication

1) Inductioneg HDACis

2) Immune modulation/eradicationeg Thx vaccine, anti-PD1

3) Therapy intensification?eg, replication in reservoirs (gut, brain?), persistent antigen production

Other approaches?

Potential Modalities and Timelines

Now– Induction therapy: SAHA– Negative Regulators: anti-PD1– Immune modulators: IFN, others– Therapeutic vaccination

Future– Combination induction therapies– Antibodies to other negative regulators– Other approaches?

What do we measure and how do we claim success?

How do we test this hypothesis?From the test tube to humans

In vitro tools Compound or Target identification

Cell-based assays, siRNAs

Compounds/drug leads (One or more MOA?)

Animal modelsPD markers; Efficacy

Human studiesBiomarkers/clinical surrogates

HIV-1 Latency Pre-clinical In Vivo Models

Animal models are critical for understanding viral persistence and testing novel concepts– Can model HAART in HIV-infected humans (eg, RTIs and InSTIs)– Time of infection & HAART initiation can be standardized.– It is possible to evaluate reservoirs in tissues eg, GALT & CNS– Viral rebound as an critical endpoint can be monitored.

– Rodent and Macaque Models

Approaches that delay or decrease viral rebound can provide information for the design of novel and/or combination clinical trials and help validate surrogate endpoints.

Is it the same in all patients?

Time from infection (acute vs chronic)– Initiation of therapy and nadir CD4

Route of infectionAgeGenetic factors, including

– Race– Ethnicity– Gender

ARV regimenOther, eg., co-infection with HCV, HCMV etc.

%T

CM c

ells

am

ong

HIV

+ c

ells

= 0.66p = 0.004

%T

TM

+E

M c

ells

am

ong

HIV

+ c

ells

= -0.64p = 0.006

CD4 count (cells/µl) CD4 count (cells/µl)200 700 1200

0

20

40

60

80

100

200 700 12000

20

40

60

80

100

TCM contribution TTM contribution

The HIV Reservoir Varies with CD4 Count

Courtesy of N. Chomont

Summary and Outstanding Issues

Multiple and perhaps “inter-dependent” processes contribute to the inability to eradicate HIV with ARV therapy– Will any one approach be sufficient for eradication? – Will the same combination of interventions work for all patients?

Various interventions which can address at least some of these issues are being explored including small molecules which may activate latent viral gene expression– Activators can manifest differential activity in different cell based assays; do these

differences reflect a spectrum of biologically relevant mechanisms?– HDACIs appear to be among the most “robust”, may provide an anchor for

combinations?– Will activation therapy be sufficient without modulation of the immune response?– Can the immune response be modulated without blocking persistent viremia?

Evaluating approaches individually and in combination in well validated animal models will be critical to understand many of these issues– What model or models will most accurately reflect HIV-1 persistence

(reservoirs/sanctuaries depend on virus, species, immune response, choice of ART)? – What data is required to justify clinical evaluation?– What surrogates can be used to provide an early signal of efficacy and would be

sufficiently robust to trigger therapy interruption?

Acknowledgements

Amy EspesethMarta MajdanCamil SayeghChris Tan

Collaborators: David Margolis Una O’Doherty Doug Richman Jeff Lifson Paul Luciw Tom North Warner Greene Eric Verdin

Many others