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July 2010
Can we eradicate HIV…What do we need to answer the question?
Daria HazudaMerck 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?
Residual replication•Sanctuaries; drug penetration•Efficacy, cell type differences
Persistent HIV expression•Replication competent?•Immune disfunction?
Latently infected cells
InflammationHomeostatic Proliferation
These are not mutually exclusive mechanisms; will multiple approaches be required?
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.
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•Efficacy, cell type differences
Persistent HIV expression•Replication competent?
Latently infected cells
InflammationHomeostatic Proliferation
These are not mutually exclusive mechanisms; will multiple approaches be required?
Rationale 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
Functional Cure
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
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
Where are we now…
HIV LatencyCell Culture Models
Integrated LTR-reporter constructs– Advantages: LTR is inducible by compounds that activate latent HIV,
amenable to siRNA screening– Disadvantages: highly reductionist system
Chronically infected, inducible cell lines– Advantages: complete integrated HIV genome– Disadvantages: clonal, each line has a single integration site, some
have defective Tat/Tar
Retroviral vectors – Advantages: GFP reporters allow sorting of population of transduced
cells, mixed population– Disadvantages: Constructs integrated into heterochromatin; HIV is
more likely to integrate into transcribed genes (mixed population
HIV LatencyMore Complex Cell Culture Systems
Ex-vivo infected primary resting CD4+ T cells– minimal LTR reporters
LGIT – HIV-1 provirus
Resting CD4+ T cells Bcl-2-transduced resting CD4+ T cells
Resting CD4+ T cells isolated from HIV+ aviremic patients– Quantification of viral DNA/RNA in memory T cell subsets – Viral outgrowth assays
How do they compare? Which is most biologically relevant?
Merck High Thoughput Screen Assay for Activators of Latent HIV-1 Gene Expression
24 hours
Add Compounds
Hela P4/R5 cells
24 hours
-Galactosidase Assay
E.coli lacZHIV LTR polyA
-galactosidaseUninfected cell
E.coli lacZHIV LTR polyA
- 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 of
LTR-LTR-Gal HTSGal HTS
NFAT-BLA Jurkat cell assayNFAT-BLA Jurkat cell assay
HDAC activity assay (novel HDACIs)HDAC activity assay (novel HDACIs)
ToxicityToxicityChemical attractivenessChemical attractivenessFurther chacterization eg ACH-2, J1.1, primary cells, ex vivoFurther chacterization eg ACH-2, J1.1, primary cells, ex vivo
Non-mechanism based screening can identify novel HIV-1 activators
Characterizing 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
HDACinhibition
NFκBactivation
pTEFbrelease
ClinicalSamples^
Ex-vivoCD4 T cells*
ProvirusJurkat**
LBITJurkat#
LTR-BgalHeLa#
VPA + +/- + + -TSA +/- + + +
HMBA +/- + + +
prostratin + + + +TNFa +/- + + -
novel - MERCK - + - - ++/-
Are these meaningful differences or a reflection of the spectrum of relevant biology?
Proposed mechanisms to affect latent proviral HIV-1 expression
Adapted from Richman et al, 2009
Me
MTInhibitor
MT
MeMT
Potential HIV-1 Latency Activation Therapies
Histone deacetylase (HDAC) inhibitors– Class I-selective: SAHA, others (MRL)– Non-selective: Trichostatin A (TSA), valproic acid (VPA)
NF-kB activators– Prostratin, PMA, TNFa
Akt/HEXIM-1 modulators– Hexamethylbisacetamide (HMBA)
Histone methyltransferase (HMT) inhibitors– DZNep: targets Ezh2 (trimethylates H3-K27/H4-K20)– Chaetocin: targets su(var)3-9 (methylates H3-K9)
Jak/Stat pathway– IL-7
Lessons from Oncology: Synergy with HDACIs
16
Synergistic reactivation of latent HIV-1Synergistic reactivation of latent HIV-1 Synergistic reactivation using combinations of agonists demonstrated in cell lines
Suggested as a potentially more robust approach to reactivate HIV-1 derived from various patients, viral subtypes, or LTR mutants
Low dose may result in diminished AEs in clinic
U1 cells J-Lat cells
Reuse et al, 2009
Burnett et al, 2010
SAHA in combination with MRL HIV “inducing” compounds synergize to activate the HIV LTR
1.40%
L127 1M
1.76%
L412 2.5 M
L495 2.5M
1.21%4.09%
L801 1M
SAHA +L127
1.89% 2.87%
SAHA +L412
SAHA +L495
73.3%
SAHA +L801
76.6%
1.21%
Untreated
SAHA 250nM
1.89%
Controls Compounds alone Compounds + SAHA
J89 Cells: latently infected Jurkat cell line with a single integrated copy of the HIV genome with EGFP as a marker for HIV expression upon stimulation of the LTR. METHODS: J89 cells were incubated with DMSO, L127, L412, L495 or L801 +/- SAHA for 19hrs. GFP expression was measured by flow cytometry. (Archin, et al unpublished)
A alone B alone A + SAHA B + SAHA
C alone D alone C + SAHA D + SAHA
Can Activation Alone “purge” the Reservoir?
Latent Cells Immune
HDACIs + ??? OR Modulator?
Activated cells
Can Activation Alone “purge” the Reservoir?
Latent Cells Immune HDACIs + ??? plus Modulator?
Activated cellsThX
VaccineImmunotoxin, anti-PD1, etc
Can you “reset” the immune system without therapy intensification and shutting off persistent antigen production?
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)– Parameters such as time of infection and HAART initiation can be
standardized.– It is possible to extensively evaluate reservoirs in tissues eg, GALT & CNS– Viral rebound as an critical endpoint can be monitored.
Rodent Models – SCID-hu mouse (human transplants of thymus, fetal liver or PBMCs– BLT mouse (human bone marrow, liver, thymus)
More complete systemic reconstitution of all major human hematopoetic lineages including T, B, monocyte/macrophage, dendritic and NK cells.
Macaque Models: SIV, SHIV etc
Approaches that delay or decrease viral rebound can provide information for the design of novel and/or combination strategies.
RT-SHIV Study: HAART and Induction w/HDACI & PKC activator(Paul Luciw, et al unpublished)
6 wks 32 - 35 wks 8 wks 1wk
RT-SHIVInoculation
HAART(FTC + PMPA + Efavirenz)
HAART + Induction
Necropsy
Rebound (16 wks)No treatments
2 wks: 5 cycles 6 wks: 1 cycle per wk
Weekly/biweekly analysis: plasma viral RNA, CBC, FACS
Necropsy
0 5 10 15 20 25 30 350
1
2
3
4
5
6
7
8 36160
3625336348
3634936488
3654436661
Start Treatment
3635336166
Weeks Post Infection
Log
vRN
A C
opie
s/m
lLongitudinal Analysis of Viral Loads During HAART• Low-Level Viremia Persists Despite Effective HAART
Fig.3A
Treatment HAART + Induction + Rebound HAART + Rebound
MonkeyMMU 36160
MMU 36349
MMU 36488
MMU 36353
MMU 36544
MMU 36913
MMU 35685
MMU 35940
MMU 36098
A. Resting lymphoid tissues Axillary LN 53 1 1 1,100 26,000 92,000 3,000 10,000 350 Inguinal LN 250 68 45 3,200 54,000 25,000 23,000 8,700 2,700 Iliac LN 30 0 44 3,500 42,000 2,400 48,000 27,000 <5 Obturator LN 530 <1 6 550 90,000 ND ND ND NDB. Active lymphoid tissues
Mesenteric LN 3 5 21 4 41,000 810 6,100 31,000 43 Cervical LN 1 <1 <1 <2 43,000 330 13 880 <1 Tonsil 520 0 430 3 13,000 69,000 47,000 360 440C. Primary lymphoid organs Spleen 110 1 2 20 130,000 59,000 11,000 25,000 83 Thymus 0 <1 <1 <3 <1 710 <1 46 <1 Bone marrow <1 <1 <1 <8 18 <2 67 <1 <1D. GI tract tissues Duodenum <1 <1 <1 1 2,100 830 <5 <2 55 Jejunum <1 68 <2 2 6,400 <3 230 15 15 Ileum <1 <1 <1 3 18,000 820 <4 <2 <1 Cecum 7 <1 1 1 4,600 2,700 <1 220 <2 Colon 61 <1 <4 3 9,900 580 610 38,000 140E. Other Liver 1 <1 <2 <1 150 47 ND ND ND Heart <1 <1 <2 <1 30 <1 ND ND ND Lung <2 <1 <2 <1 330 <1 ND ND NDPlasma vRNA copies /ml 316 <50 <50 1850 1,292,699 916 179 1,238 <50
Tissue vRNA levels are reduced following combination induction treatment and rebound
TreatmentHAART + Induction + Rebound
HAART + Rebound
Monkey MMU 36160MMU 36349MMU 36488
MMU 36353
MMU36544
MMU 36913
MMU 35685
MMU 35940
MMU 36098
A. Resting lymphoid tissues
Axillary LN 230 180 640 2,000 2,500 1,200 140 <1 130
Inguinal LN 150 100 450 4,600 2,500 6,000 1,100 930 1,200
Iliac LN 170 140 61 730 6,100 27 400 670 <5
Obturator LN 400 160 97 1,800 4,500 ND ND ND NDB. Active lymphoid tissues
Mesenteric LN 550 170 720 1,700 2,200 6,400 340 1,500 760
Cervical LN 9 54 37 59 6,100 290 <15 830 950
Tonsil 100 9 110 150 6,000 2,800 3,900 1,900 570C. Primary lymphoid organs
Spleen 110 15 110 650 12,000 680 74 660 180
Thymus 6 <1 <1 13 44 16 51 <1 <1
Bone marrow <1 <1 <1 <8 8 15 110 <1 <!D. GI tract tissues
Duodenum 3 19 99 140 2,400 110 <4 <1 190
Jejunum 15 37 49 76 610 140 520 200 69
Ileum <2 14 70 210 830 69 <3 <1 <1
Cecum 85 5 3 130 1,300 3,200 2 170 <2
Colon 440 84 450 61 2,700 <1 ND ND 200E. Other
Liver <1 <2 110 <1 52 <1 ND ND ND
Heart 19 <1 <2 <1 58 <1 ND ND ND
Lung 55 25 5 13 510 7 ND ND ND
6. Plasma vRNA copies/ml 316 <50 <50 1850 1,292,699 916 179 1,238 <50
Tissue vDNA levels are reduced following combination induction treatment and rebound
No Difference in rebound plasma viremia after discontinuation of HAART and inducers
Group 1B = HAART plus induction; Group 3 = HAART only
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; Are these differences biologically meaningful or reflect a specturm of biological mechanisms relevant in “non-uniform” systems?
– HDACIs appear to be the most robust, provide an anchor for combinations?– Will activation therapy be sufficient without modulation of the immune response;
can the immune response be modulated wiithout blocking pesistent viremia? Evaluating these approaches individually and in combination in well validated
animal models will be critical to understand many of these issues– What data will provide sufficient evidence to justify clinical evaluation?– What clinical 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
Tissue Fold95% Confidence
Interval
Group Outcome Estimate Lower Upper P-value
A Resting lymphoid vRNA 185 3.2 10806 0.015
B Chronic, active lymphoid vRNA 446 17.1 11648 0.0011
C Primary lymphoid vRNA 34 2.9 391 0.0078
D GI tract vRNA 151 18.5 1236 <0.0001
A-D vRNA 143 11.9 1706 0.0001
A Resting lymphoid vDNA 2.3 0.38 13.8 0.35
B Chronic, active lymphoid vDNA 13.9 3.7 52.2 0.0006
C Primary lymphoid vDNA 9.2 0.99 84.3 0.051
D GI tract vDNA 4.0 0.58 27.8 0.15
A-D vDNA 5.4 1.14 25.6 0.034
Statistical analysis comparing vRNA and vDNA tissue levels between induction
and non-induction groups