Fifth Annual Mitchell Memorial Lecture, October 6, 2014, at UC San Diego, featuring Dr. Jonathan Karn of Case Western Reserve University speaking on "Lessons Learned from models for HIV latency helping to formulate virus eradication strategies."

1. 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

2. AIDS in the US: The Memorial Quilt
People visit the AIDS Memorial Quilt on display on the National
Mall in Washington, on Thursday, July 5, 2012.
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. 3
Why Attempt to Eradicate HIV Infections?
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

4. Well suppressed patients rebound when HAART is stopped
x

5. Why can’t we cure HIV infections with HAART?
Residual replication
Sanctuary sites
+
Poor drug penetration Latently infected cells
Memory T-cells
Microglial cells
Macrophages
Homeostatic
Proliferation
Chronic inflammation
Stem cell infection

6. 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”)

7. Tickling the tail of the dragon: The “shock and kill”
Other Challenges:
•!Clearance of infected cells
•!Clearance of virions
Other •!Complete block of new infection
•!Clearance •!Clearance •!Complete approach
Anti-latency
therapy
Immunological
enhancement
Other Challenges:
Clearance of infected cells
Clearance of virions
Complete block of new infection

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

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

10. Specific integration sites
and orientations of
proviruses in different
Jurkat T-cell lines

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

12. Tat is undetectable in latently infected cells

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

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

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

16. 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

17. EpMod (Transomics Epigenetic Modifier Lenti Virus)
shRNA library screening in JC2mC cell lines
Unsorted 2nd Sort 4th Sort
30
25
20
15
10
5
0
Control
(mC only)
at 1st
Sort (G+
& mC+)
at 2nd
Sort (G+
& mC+)
at 3rd
Sort (G+
& mC+)
at 4th
Sort (G+
& mC+)
% GFP+
Enrichment of re-activated cells
mCherry (HIV-1)
GFP (shRNAs)

18. 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

19. Epigenetic silencing of HIV is progressive and hierarchical

20. Primary cell models: Reporter and polarization condition

21. T-cell differentiation pathways

22. Confirmation of polarization phenotypes:
Transcription factors

23. Induced entry into latency through cytokine restriction

24. CycD3 and CycB1 provide objective markers for T-cell quiescence

25. The CD8a-GFP fusion protein persists in quiescent cells

26. Activation of P-TEFb in resting memory cells

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

28. SP1-TE7Fb5 a ssembly in resting memory T-cells following
T-cell receptor stimulation

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

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

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

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

33. TGF-b and JAK-STAT pathways help maintain quiescence

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

35. Knockdown of ESR1 does not activate NF-kB

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

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

38. 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

39. Inducible RNA assay demonstrates latency in cells
from untreated patients

40. 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

41. Downregulation of MHC in latently infected cells induced by SAHA

42. NK cell surveillance assay
Naïve CD4
T cells
Donor PBMC
Polarized
to Th1
Infected PHR
CD8a-GFP
Quiescent
condition
CD8a
isolation
Mix pop
Uninfected
cells
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)

43. Sensitive detection of NK-mediated killing using Pantoxilux G2D2
assay (Oncolmmunin)
Apoptosis
Afonina. 2010. Immunol
Reviews

44. NK cells also kill other latently infected
T cell subsets after HIV reactivation with Panob/Bryo
Minus NK +IL21 NK (1:1)
NK cells were
activated with
feeder cells
expressing
membrane-bound
IL-21
Th1
64%
93% 7% 36%
0 101 102 103 104 105
YG582-A
0 101 102 103 104 105
B530-A
36.2% 64.3%
Th2
Treg
90% 10% 28% 72%
0 101 102 103 104 105
YG582-A
0 101 102 103 104 105
B530-A
28.0% 72.4%
85% 15% 36% 64%
0 101 102 103 104 105
YG582-A
0 101 102 103 104 105
B530-A
35.7% 65.5%
0 101 102 103 104 105
PS cleavage
YG582-A
0 101 102 103 104 105
B530-A
92.7% 7.4%
0 101 102 103 104 105
YG582-A
0 101 102 103 104 105
B530-A
90.4% 9.7%
0 101 102 103 104 105
YG582-A
0 101 102 103 104 105
B530-A
85.1% 15.0%
+ Panobinostat
+ Bryostatin

45. A hybrid approach: Genetically engineered NK-cell killing
Lymphocyte
expansion
Infusion of
NK cells
timed with
HIV induction
Engineered NK-cells
reach tissues
with activated HIV-infected
cells
Killing of
targeted
cells
Insertion
of CAR
Blood
drawn

46. Challenges for developing a safe and effective “Shock”
• 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!
Developing effective killing strategies will be
even harder!

47. 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?

48. 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)