18 th Annual Conference of the British HIV Association (BHIVA)

Professor Mario Stevenson University of Miami Leonard M Miller School of Medicine, Florida, USA

18-20 April 2012, The International Convention Centre, Birmingham

18 th Annual Conference of the British HIV Association (BHIVA)

Professor Mario Stevenson University of Miami Leonard M Miller School of Medicine, Florida, USA

COMPETING INTEREST OF FINANCIAL VALUE > £1,000: Speaker Name Statement

Professor Mario None Stevenson:

Date April 2012

18-20 April 2012, The International Convention Centre, Birmingham

1 ART now, ART to come: Is there a need for new ARVs?

BHIVA, Birmingham, April 2012.

Mario Stevenson, Ph.D. University of Miami Miller School of Medicine.

HAART IS EFFECTIVE:

• Rapidly achieves 4-6 log reduction in viremia to below detectable limits. • Suppression to below detectable levels is rapid and durable.

2 What sustains HIV-1 in the face of HAART?

3 What accounts for the extreme persistence of the viral reservoir ?- predictions

• HAART stops active replication. Latency allows lifelong persistence of the . – Prediction, intensification won’t change anything. • Incomplete virologic suppression creates conditions for reservoir replenishment. – Prediction, different treatments may perturb the viral reservoirs.

How do we probe the viral reservoirs that persist in the face of HAART?

• New classes of antiretroviral agents widen treatment options for infected patients but also offer reagents with which to probe the reservoirs that persist in the face of HAART.

4 Monitoring the impact of therapy intensification:

• INTegRAL study in collaboration with J. Picado & B. Clotet: impact of Raltegravir intensification. • 69 patients on suppressive 3 drug HAART regimen randomized to intensify with Raltegravir (n=44) or to continue HAART (n=24). • Episomal cDNA and immune activation parameters were monitored.

Cytoplasm

Nucleus

5 Cytoplasm 

 Raltegravir  Nucleus

Episomes increase upon Raltegravir intensification.

Buzon et al., Nature Med. 2010

6 Increase in episomes following Raltegravir intensification can Cytoplasm only be explained  by de novo infection.

 Raltegravir  Nucleus

Reservoirs of cryptic replication?

• If treatment intensification impacts the viral reservoir, does this indicate incomplete suppression? • Some tissue reservoirs may not reflect what is happening in the blood. • Do these reservoirs constitute sanctuary sites for viral replication? • Attention has focused on the lymphoid tissue in the gut since it is the predominant site of viral replication.

7 Reservoirs of HIV-1 persistence in HAART

M. Stevenson, Scientific American 2008

The role of lymphoid tissue in viral persistence.

• PO1 collaboration to identify the virologic response in tissue to ART. • Baseline and sequential sampling over 6 months of gut and lymph node from patients initiating suppressive ART. • Examination for viral infection intermediates (episomal, proviral, unintegrated linear cDNA ) cell associated RNA and intracellular drug levels.

8 Cytoplasm ~90%

~10% Nucleus

Results.

• Decay dynamics of viral replication intermediates follow 2 patterns: - Patients whose cDNA levels fall to below detectable by 6 months. - Patients whose cDNA levels at 6 months in 1 or more tissue compartments remain detectable or increase over entry-point or 1 month levels.

9 Results.

• Decay dynamics of viral replication intermediates follow 2 patterns: - Patients whose cDNA levels fall to below detectable by 6 months (3/10) - Patients whose cDNA levels at 6 months in 1 or more tissue compartments remain detectable or increase over entry-point or 1 month levels.

Results.

• Decay dynamics of viral replication intermediates follow 2 patterns: - Patients whose cDNA levels fall to below detectable by 6 months. - Patients whose cDNA levels at 6 months in 1 or more tissue compartments remain detectable or increase over entry-point or 1 month levels (7/10).

10 Basis for incomplete virologic response in lymphoid compartments:

• Does a suboptimal virologic response in tissue occur in the face of effective drug sequestration in the tissue? • Determine intracellular drug levels relative to concentrations in peripheral blood lymphocytes.

Improving virologic response in tissue reservoirs: • Despite suppressive HAART , de novo viral infection persists in lymphoid tissue (esp. LN). • Inability to sequester antivirals in tissue lymphocytes may create conditions for replenishment of the viral reservoirs. • These findings have implications for strategies aimed at viral control

11 New Therapeutic Targets:

Harnessing Cellular Restrictions.

Retrovirus:Host interactions Courtesy Paul Bieniasz

Infected Cell Target Cell Release

Entry

Reverse transcription Envelope Nuclear import integration

Assembly

viral RNA, Gag and Pol

12 :Host interactions Courtesy Paul Bieniasz

Infected Cell Target Cell Release

Tetherin restricts virus particle release Entry

Reverse transcription Envelope protein Nuclear import integration

Assembly

viral RNA, Gag and Pol proteins

Retrovirus:Host interactions Courtesy Paul Bieniasz

Infected Cell Target Cell Release

Tetherin restricts virus particle release Entry

Apobec3 restricts cDNA synthesis/integrity Reverse transcription Envelope protein Nuclear import integration

Assembly

viral RNA, Gag and Pol proteins

13 If our cells carry such potent restrictions, why are we still susceptible to HIV-1 infection?

Vif inhibits APOBEC3G by inducing proteasomeproteasome--mediatedmediated degradation

Mariani et al. , Stopak et al., Sheehy et al., Marin et al., Conticello et al., Yu et al., Mehle et al.

14 Cell-based screen for Vif antagonists A C Vif ∆∆∆ Vif Vif 1 2 34 5 6 78 9 10 11 12 12 3 45 6 78 9 10 1112 1 2 34 5 6 78 9 1011 12 A A A A3G YFP B B B A3G YFP C C C A3G YFP D D D E E E A3G YFP F F F A3G YFP G G G H H H A3G YFP A3G YFP

1 2 34 5 6 7 89101112

A

B

C

D

YFP Intensity 1 00 E 80

60 F 40

20 G 0 Vif :APO-YFP Vif:APO-YFP+Drug d VIF:APO-YFP H

30,000 small molecules B DMSO small molecule library DMSO D 1 2 3 4 5 6 7 8 9 10 11 12 (library) A B C D 537 small molecules E F (primary screen) G H

Vif + A3G-YFP ∆ vif + A3G-YFP 66 small molecules (secondary screen)

Validation of RN18 as vif antagonist:

• Since vif is required only in cells that express Apobec3, a vif antagonist would be predicted only to exert antiviral activity in non-permissive cells (i.e. in cells that express Apobec3).

15 Non-Permissive A B E

H9 H9 CEM 30000 35000 120000 25000 30000 50 50 100000 y y t y t i 25000 i 20000 25 25 t i v v 80000 i i v t i t 20000 10 t c 10 LAI wt 15000 c c 60000 A A

A LAI vif- 5 15000 5

T T 10000 T 40000

R 1 10000

R 1 R 5000 0 5000 0 20000 0 0 0 3 6 10 12 14 3 6 10 12 14 3 5 7 9 11 14 Days post-infection Days post-infection Days post-infection

Permissive C D F MT-4 CEM-SS MT-4 60000 120000 45000 50000 40000 100000 50 35000

y 50 t

40000 y i

25 t

i 80000 y v 25 30000 i t i v t i v

10 t i c

30000 10 t 25000 c c A 60000

5 A LAI wt A

T 5 20000 T

T LAI vif-

R 20000 1 R 40000 1 R 15000 0 0 10000 20000 10000 5000 0 0 3 6 10 12 14 3 6 10 12 14 0 Days post-infection Days post-infection 3 5 7 9 11 14 Days post-infection

RN18-resistance profiles.

• Experience with other classes of ARVs predicts that acquiring resistance to an agent will also exhibit resistance to related compounds. • Examine sensitivity of RN18-resistant virus to RN18 analogs

16 RN18-resistant virus retains sensitivity to some RN18 analogs

IMA53 versus WT virus IMA53 versus RN18-resist. virus 16000 16000 14000 14000 12000 ima53/50uM 12000 ima53/50uM 10000 ima53/25uM 10000 8000 ima53/25uM ima53/10uM 8000 6000 ima53/10uM 6000 RT cpm/5uL RT 4000 ima53/5uM RT cpm/5uL RT 4000 ima53/5uM 2000 ima53/1uM 0 2000 ima53/1uM Control 0 5 10 15 0 Control days p.i. 0 5 10 15 days p.i.

IMC15 versus WT virus IMC15 versus RN18-resist. virus 18000 18000 16000 16000 14000 14000 imc15/50uM imc15/50uM 12000 12000 imc15/25uM 10000 imc15/25uM 10000 8000 8000 imc15/10uM imc15/10uM 6000 6000 cpm/5uL RT imc15/5uM RT cpm/5uL RT imc15/5uM 4000 4000 imc15/1uM 2000 imc15/1uM 2000 0 Control 0 Control 0 5 10 15 0 5 10 15 days p.i. days p.i.

Structure Activity Relationship on RN18: conclusions • SAR on approx. 500 analogs of RN18 • SAR has identified active compounds with superior numbers that are drug like molecules. • Novel resistance profiles: virus resistant to RN18 remains sensitive to its analogs.

17 Development of small-molecule Vif antagonists:

RN18 is a bona fide vif antagonist -restricts viral replication only in the presence of its target and increases the extent of deamination in viral cDNA (Nathans et al., Nature Biotech.) Proof-of-principle evidence that cellular restrictions can be harnessed as novel antivirals. RN18 is a drug-like molecule. Preclinical development of these novel antagonists is underway (NIH UO1 and Industry support)

Viral Innate cellular countermeasures defenses (accessory genes)

18 Accessory Gene inhibitors

Cellular antagonists

Collaborators:

IRSI Caixa, Barcelona Burnham Institute Ventura Clotet Tariq Rana Javier Picado Roger Paredes Vaccine Research Center, NIH University of Minnesota Danny Douek Tim Schacker Ashley Haase Cavan Reilly UCSF University of Nebraska Steve Deeks Courtney Fletcher Mike McCune

19