Lessons from models for HIV latency helping to formulate virus eradication

strategies Jonathan Karn

Rick Mitchell Memorial Lecture University of California San Diego

San Diego, CA 6 October 2014

People visit the AIDS Memorial Quilt on display on the National Mall in Washington, on Thursday, July 5, 2012.

AIDS in the US: The Memorial Quilt

Ø  The HIV epidemic has claimed more than 575,000 lives

Ø  The CDC estimates that there are from 500,000 to 1.1 million individuals living with HIV

Ø  Nearly 18,000 AIDS patients die each year

Ø  Around 56,000 new HIV infections are reported annually

Every 9 minutes and 30 seconds someone is infected with HIV

3

Ø  HAART is an expensive treatment requiring careful monitoring

Ø  Patients want to be free of drugs – removes stigmas

Ø  Poor compliance can complicate treatment and lead to resistance and transmission

Ø  Less than 20% of patients in the US are in effective therapy

Ø  Non-AIDS pathology leads to premature death

Why Attempt to Eradicate HIV Infections?

Well suppressed patients rebound when HAART is stopped

x

Homeostatic Proliferation

Chronic inflammation Stem cell infection

Latently infected cells Memory T-cells Microglial cells Macrophages

Residual replication Sanctuary sites

Poor drug penetration +

Why can’t we cure HIV infections with HAART?

Current Strategies

•  Start ART very early before reservoirs are fully established or intensify treatment

•  Eliminate residual virus replication by ART intensification

•  Replace the immune system with cells engineered to be “resistant” to HIV (CCR5-)

•  Induce proviruses and then eliminate latently infected cells (“Shock and kill”)

Other Challenges: •!Clearance of infected cells

•!Clearance of virions

•!Complete block of new infection

Other Challenges: •!Clearance of infected cells

•!Clearance of virions

•!Complete block of new infection

Tickling the tail of the dragon: The “shock and kill” approach

Anti-latency therapy

Immunological enhancement

Other Challenges: •!Clearance of infected cells

•!Clearance of virions

•!Complete block of new infection

Disruption of the Tat/P-TEFb feedback leads to HIV proviral latency

Induction of HIV transcription in clones of latently infected T-cells is strictly dependent upon NF-κB

Specific integration sites and orientations of

proviruses in different Jurkat T-cell lines

RNAP II levels at the HIV LTR fluctuate in parallel to nuclear NF-κB levels after TNF-α induction

Tat is undetectable in latently infected cells

ChIP-seq assays show RNA polymerase is paused downstream of the transcription start site in latent and induced proviruses

RNAP II in latent cells is paused at the 5’ LTR

Reversal of latency involves chromatin remodeling and Tat/P-TEFb/SEC complex recruitment

Strategy for identifying a comprehensive set of latency factors using shRNA libraries

Cellecta: Total 82,500 shRNAs targeting 15,439 genes with shRNA/gene 4 to 6

EpiMod: Total 4186 shRNAs targeting 407 genes with shRNA/

gene: 6 to 15

0

5

10

15

20

25

30

Control (mC only)

at 1st Sort (G+ & mC+)

at 2nd Sort (G+ & mC+)

at 3rd Sort (G+ & mC+)

at 4th Sort (G+ & mC+)

% G

FP+

Enrichment of re-activated cells

EpMod (Transomics Epigenetic Modifier Lenti Virus) shRNA library screening in JC2mC cell lines

mC

herr

y (H

IV-1

)

GFP (shRNAs)

Unsorted 2nd Sort 4th Sort

Both PCR-1 and PCR-2 are present at the repressed LTR PCR-2 z-score EZH2 1.81 EED 2.39 SUZ12 0.28 RBBP4 1.36 RBBP7 2.26 JARID2 4.50 PCR-1 EZH1 3.28 CBX2 6.86 CBX4 1.50 CBX6 1.43 CBX8 2.71 PHC1 2.96 PHC2 2.84 PHC3 6.60 PCGF1 1.37 PCGF2 1.05 BMI1 6.54 RNF2 1.69

PCR-1 related z-score L3MBTL3 21.28 PSIP1 12.28

trxG z-score RBBP5 1.75 ASH2L 2.45 MLL2 1.11 MLL3 4.05 MLL4 2.13

Epigenetic silencing of HIV is progressive and hierarchical

Primary cell models: Reporter and polarization condition

T-cell differentiation pathways

Confirmation of polarization phenotypes: Transcription factors

Induced entry into latency through cytokine restriction

CycD3 and CycB1 provide objective markers for T-cell quiescence

The CD8a-GFP fusion protein persists in quiescent cells

Activation of P-TEFb in resting memory cells

Phosphorylation of the T-loop of CDK9 regulates P-TEFb

S175 P-TEFb assembly in resting memory T-cells following T-cell receptor stimulation

The pS175 marker correlates well with transcriptional activation of the latent provirus

Activation of P-TEFb in latently infected Th17 cells strictly correlates with proviral reactivation

RNA polymerase is paused downstream of the transcription start site in latent and induced proviruses in primary cells

RNA seq reveals program of cellular gene responses during Th17 and Treg cell reactivation

TGF-b and JAK-STAT pathways help maintain quiescence

ESR1 is a central mediator of HIV latency in Jurkat T-cells

Knockdown of ESR1 does not activate NF-kB

Blocking of ESR-1 and its upstream modulator SRC3 re-activates latent HIV-1 provirus in primary T-cells

Exchange of repressors and activators during transcriptional activation of HIV-1

From a molecular perspective latency is an integral feature of the HIV life cycle

•  NF-κB is only needed to initiate transcription from latent proviruses

•  Transactivation can be thought of as a way to turn on and off transcription in response to changes in the cellular environment

•  10 to 20% of infections are silent integration events

•  Non-suppressed patients have latent proviruses

•  Latency is probably an escape mechanism from immune responses and thus aids virus dissemination

Inducible RNA assay demonstrates latency in cells from untreated patients

Fauci et al. Nat Rev Immunol. 2005 Release granzymes

and perforins

There may be a window of opportunity after proviral

activation when NK cells can target and kill latently infected primary T cells due to MHC

downregulation by Nef

Downregulation of MHC in latently infected cells induced by SAHA

NK cell surveillance assay

Donor PBMC

Naïve CD4 T cells

Polarized to Th1

Infected PHR CD8a-GFP

Quiescent condition

CD8a isolation

Mix pop

Expand/activate NK cells Activate NK cells with irradiated C9 (K562 expressing membrane-bound IL-21); Isolate NK cells and activate for 3 days with IL-2 (500 IU/ml)

Uninfected cells

Sensitive detection of NK-mediated killing using Pantoxilux G2D2 assay (Oncolmmunin)

Apoptosis

Afonina. 2010. Immunol Reviews

+IL21 NK (1:1) Minus NK

NK cells also kill other latently infected T cell subsets after HIV reactivation with Panob/Bryo

NK cells were activated with feeder cells expressing membrane-bound IL-21

Th1

0 101 102 103 104 105

YG582-A

010

110

210

310

410

5B530-A

36.2% 64.3%

Th2

Treg

0 101 102 103 104 105

YG582-A

010

110

210

310

410

5B530-A

28.0% 72.4%

0 101 102 103 104 105

YG582-A

010

110

210

310

410

5B530-A

35.7% 65.5%

64%

PS cleavage

0 101 102 103 104 105

YG582-A010

110

210

310

410

5B530-A

92.7% 7.4%

0 101 102 103 104 105

YG582-A

010

110

210

310

410

5B530-A

90.4% 9.7%

0 101 102 103 104 105

YG582-A

010

110

210

310

410

5B530-A

85.1% 15.0%

93% 7% 36%

72% 90% 10% 28%

64% 85% 15% 36%

+ P

anob

inos

tat

+ B

ryos

tatin

A hybrid approach: Genetically engineered NK-cell killing

Engineered NK-cells reach tissues with activated HIV-

infected cells

Killing of targeted

cells

Infusion of NK cells

timed with HIV induction

Insertion of CAR

Lymphocyte expansion

Blood drawn

•  Identifying a shock agent won’t be easy, however, a wide set of factors is being identified.

•  Synergy between inducers of P-TEFb and factors reversing epigenetic silencing is biologically plausible and likely to yield the best compound combinations.

•  Primary cell models for latency are still imperfect, although they are getting better, and need to embrace a full range of cell types including myeloid cells.

•  Don’t forget the brain: The virus may be lurking in more than one place not only the “fashionable” T-cells!

Challenges for developing a safe and effective “Shock”

Developing effective killing strategies will be even harder!

Is HIV eradication practical and worthwhile?

Ø  With advances in HIV therapy, is striving for HIV eradication in more than a few specific cases worth the drastic interventions likely to be required to accomplish this?

Ø  Will life-long therapy only be replaced by life-long monitoring?

Ø  Will it be cost-effective?

Ø  Are there simpler approaches?

Ø  Will studying latency lead to improved care even if eradication is unfeasible?

Current Laboratory Members

Curtis Dobrowolski (Th17) Uri Mbonye (P-TEFb)

Biswajit Das (Screens) Kien Nguyen (Epigenetics)

Hongxia Mao (Nef) Mary Ann Checkley (NK cells)

Michael Greenberg (Transcription)

David Alvarez (NeuroAIDS) Yoevlis Garcia (NeuroAIDS)

Stephanie Milne (NeuroAIDS)