Towards an HIV cure:

understanding the major

barriers

Sharon R LewinDirector, Doherty Institute for Infection and Immunity, The

University of Melbourne and Royal Melbourne Hospital and

Consultant physician, The Alfred Hospital, Melbourne, Australia

1st APACC Conference, Hong Kong, China, May 17-19, 2016

Only one case of HIV cure:

Timothy Brown

Outline: major barriers to cure

HIV persistence

– HIV latency

– HIV persistence in tissues

Impaired immune function

– HIV-specific T-cell function

– Inflammation

Measuring virus persistence and biomarkers of

functional cure

HIV persistence on ART: many diverse forms

Chun TW, Nature Immunol 2015

HIV latency

Establishing HIV latency

Activated CD4+ T-cell

Resting CD4+ T-cell

Pre-activation latency

Post-activation latency

Saleh et al., Blood 2007; Cameron et al., Proc Natl Acad Sci 2010;107(39):16934-9; Evans et al Plos

Pathogens 2013; 9:12; e1003799; Shen A et al., J Virology 2013 Sep;87(17):9768-79

TCR

1

Integration

Translation 5

Host miRNA

US

RNA export 4TF

Transcription

factors

3

CoAct

Transcription

2

RNAP

MS

HIV

proteins

Rev

6

Tat

PD-1, CTLA-4

Multiple molecular factors maintain HIV

latency

Deeks S, Lewin SR et al., Nature Med 2016 (in press)

Reversing HIV latency: shock and kill

HIV DNA

HIV US RNA

HIV DNA

HIV proteins

HIV virions

Latent infection

“shock”

Cell death

Many drugs activate latency in vivo but

none eliminate latently infected cells

Latency

reversing agent

Site of action HIV latency US HIV

RNA

Plasma

RNA

HIV

DNA

Vorinostat HDACi Single dose1

Intermittent2

Continuous3

↑ ↔ ↔

Panobinostat HDACi Intermittent

dose4 ↑ +/- ↔

Romidepsin HDACi Weekly

dose5 ↑↑ ↑↑ ↔

Disulfiram AKT activation High dose

2g/day6 ↑ ↑ ↔

Bryostatin PKC agonist Low dose

10-20ug/m2 ↔ ↔ ↔

1 Archin et al., Nature 2012; 2 Archin et al., J Infect Dis 2014; 3Elliott J et al., Plos Pathogens 2014; 4Rasmussen

et al., Lancet HIV 2014; 5Sogaard et al., Plos Pathogens 2015; 6Elliott J et al., Lancet HIV 2015; 7Gutierrez et al.,

AIDS 2016

Enhancing potency of latency reversing

agents: combination activation

1 Bouchat EMBO Mol Med 2016; 2 Bouchat AIDS 2012; 3 Tripathy MK J Virol 2015; 4 Laird GG J Clin Inv 2015;5 Barnard R., HIV persistence meeting, Miami 2015; 6 Karn J, CROI 2016

Multiple synergistic combinations

HDACi + 5 aza-C (methylation inhibitor)1

HDACi + DNA methyltransferase inh2, 3

HDACi + bryostatin 4

HDACi + disulfiram 4

HDACi + screening drug library (Merck)5

HDACi + IL-156

Enhance immune mediated

killingT-cell vaccines

Antibody – bNAB, bispecific, ADCC

Reversing HIV latency: the kill

HIV DNA

HIV US RNA

HIV DNA

HIV proteins

HIV virions

Latent infection

“shock”

Cell death

“kill”

Enhance apoptosisInhibitors of apoptosis (IAP) antagonists

BCL2 antagonists

PI3k antagonists

Inhibiting BCL2 to trigger apoptosis of

latently infected cells

HIV protease

cleaves

procaspase 8 to

Casp8p41

Casp8p41 binds

BAK and induces

apoptosis

Also binds BCL2

enhances cell

survival

BCL2 antagonist

(venetoclax) in

clinical

development for

CLL (phase II/III)

Cummins et al., J Virol 2016

Patient derived cells (on

ART) treated ex vivo

with PHA/IL2+ BCL2

antagonist (venetoclax)

HIV persistence in

tissue

Chun et al. J Infect Dis 2008; Yukl et al. J Infect Dis 2010; Churchill et al. Annals Neur 2010; Fletcher et al. Proc

Natl Acad Sci 2014; Perreau et al. J Exp Med 2013

Anatomical reservoirs

Lymph node: persistence of HIV in B

cell follicles in T follicular helper cells

T follicular helper cells are highly

enriched for replication competent virus

n=9; HIV-infected individuals on ART Banga et al., CROI 2016, Boston MA

PD-1 hi cells

CXCR5+ cells

Double negative cells

Leuka

CM

X3p

R6p

Leuka

CM

X3n

R6p

Leuka

CM

X3p

R6n

Leuka

CM

X3n

R6n

1

10

100

1000

10000

Inte

gra

ted

HIV

DN

A c

op

ies

/ 10^

6 c

ells

P < 0.001

P < 0.001

P < 0.001

R6+X3+ R6+X3- R6-X3+ R6-X3-

n=20

Khoury et al., AIDS 2016; Anderson, Deeks. Lewin et al (unpublished)

Rectum: HIV is enriched in

CCR6+CXCR3+ (Th1/Th17) T-cells

Th1/Th17 Th17 Th1

X3+R6+X3-R6+X3+R6-X3-R6-

Rectum

Blood

X3+R6+X3-R6+X3+R6-X3-R6-

n=20

n=48

Blood

Brain: Microglia are infected on ART

but rare

Melissa Churchill and Lachlan Gray, Burnet Institute

In 5 patients on ART, HIV DNA detected in only one patient

CT R PNNN T RK S I H I - - GPGR A F Y T T GE I I G D I RQAH C

. . . . . . . . . . R . R . QR . . . . . . V . I . K . - . NM . . . . .

. . . . . . . . . . . . . . - - . . . . . . . . . . D . . . . . . . . . .

. . . . . . . . . . . . N . - - . . . . . L . . . . . . . . . . . . . . .

. . . . S . . . . . . . . . - - . . . . . . . . . . . . . . . . . . . . .

. R . - . . . . . . . . . . - - . R . . . . . . R . . . . . . . . . . . .

. R . . . . - . . . . . . . - - . . . . . . . . . . D . . . . . . . . . .

. R . - . . . . . . . . . . - - . R . . . . . . R . . . . . . . . E . . .

. . . . . . - . . . . . . . - - . . . . . . . . . . D . . . . . . . . . .

. I . . . . . . . . . . P . - - . . R . . . . . . E . . . . . . . . . . .

. . . . D . . S . . . . . . - - . . . . S . FA . . D V . . . . . . . . .

. . . . D . . . . . . . . . - - . . . . S . . A . . D V . . . . . . . . .

. I . . . . . . . . . . R . - - . . . Q T . . A . . D . V . . . . . . Y .

Persistent virus replication on ART in

tissue: remains controversial!

Interventions targeting tissue

Tissue site Persistent virus Target Intervention

Lymph

node

T follicular helper cell (B

cell follicle)

Disrupt follicle

Enhance migration

of CTL

Anti CD20 (NHP)

CXCL13

GI tract CCR6+CXR3+ T cells T-cell trafficking Chemokine

antagonists (NHP)

a4b7 antagonists

CNS Microglia

Astrocytes

Enhance CNS

uptake of ARV

DGV + MVC1

Residual

virus

replication

Activated T-cells

Monocyte/macrophages

Enhance tissue

uptake of ARV

Raltegravir2

Dolutegravir3

?TAF ?other

1InMIND, clinicaltrials.gov NCT02519777; 2Buzon et al., Nat Med 2010; Hatano et al., J Infect Dis 2013; 3DIORR, clinical trials.gov NCT02500446

impaired immune

function on ART

HIV persists during ART in part due to lack

of effective T cell immunity

CD8+ T-cells contribute to viral control

in SIV-infected macaques on ART

Silvestri G et al., CROI 2016, Boston MA

n=11; SIVmac239, ART=PMPA/FTC/DRV/RGV

PERIOD 1 = before CD8 depletion

PERIOD 2 = after CD8 depletion

PERIOD 3 = after CD8 reconstitution

Plasma SIV RNA

Blocking immune checkpoint markers

to boost immune function

Ex vivo blockade of PD-1, PDL-1 CTLA-4 and TIGIT enhance HIV-specific T-cells

Trautmann L, Nat Med 2006; Kaufmann D, Nat Immunol 2007; Chew GM, Plos Path 2016

Latent HIV is enriched in T-cells expressing

immune checkpoint markers

Chomont et al., Nature Med 2009

0

3

6

9

12

Rela

tive e

nrichm

ent in

inte

gra

ted H

IV D

NA Number of immune

checkpoints

1

2

3

0

Number of immune

checkpoints0 1 2 3

Expression of PD1, TIGIT,

LAG-3 or CTLA-4

Fromentin, Chomont et al., submitted

ALT-803 (IL15 superagonist) reverses

latency and boosts CTL activity ex vivo

Jones B et al., Plos Path 2016; Jones et al., Keystone meeting on HIV Persistence, April 2015

Resting CD4+ T-cells (6

billion PBMC) from patients

on ART co-cultured with

autologous CTL

ALT-803 is an IL-15 superagonist currently in phase I/II for solid organ malignancy.

Open label, single arm, dose escalation study in HIV infection planned (Schacker).

0 1 0 2 0 3 0 4 0 5 0 6 0

1 0 0

1 0 0 0

1 0 0 0 0

G A L T _ 3 8 D R C D 8 _ in tH IV

C D 3 _ C D 8 _ C D 3 8 _ H L A D R

int

HIV

GA

LT

%CD38+HLA-DR+

CD8+ T-cells

Inte

gra

ted H

IV D

NA

copie

s/ m

illio

n C

D4+

T-c

ells

0 1 0 2 0 3 0

1 0 0

1 0 0 0

1 0 0 0 0

L N _ 3 8 D R C D 8 _ in tH IV

C D 3 _ C D 8 _ C D 3 8 _ H L A D R

inth

iv d

na

% CD38+HLA-DR+

CD8+ T-cells

HIV persistence on ART correlates with

markers of activation and proliferation

Khoury, Fromentin, Chomont et al

Rectum Lymph node

Similar findings for CCR5, Ki67, PD-1, TIM3 and others in blood and tissue

p=0.023p<0.001

measuring the HIV

reservoir

Genomes producing

replication-competent virions

Gag DNA

Assay targetDetectable targets Frequency

1000

usRNA, msRNA

HIV proteins

Alu-LTR 300

30-70

?

Infectious virus 1

Viral particles 25

HIV genomes

Integrated genomes

60Intact genomesIntact genomes

Genomes producing

viral transcripts

Genomes producing

viral proteins

Genomes

producing virions

Deeks SG, Lewin SR, Chomont N et al., Nature Med 2016 (in press)

Quantifying HIV persistence

Brightfield env RNA/

gag RNA /DAPI

49%51% 52% 48%

HIV gag HIV env

Detection of HIV RNA producing cells by

flow cytometry

CEM (uninfected) : Activated ACH2 (infected)

1:100 1:1000 1:10,000 1:100,0001

10

100

1000

10000

HIV gag

HIV

en

v

Hao Lu, Doherty Institute

Detection of SIV env using CT/PET

Santangelo et al., Nature Methods 2015

Poly(ethylene glycol)-modified, 64Cu-labeled SIV Gp120–specific antibody

We need a biomarker that can predict

“cure” or “remission”

HIV DNA: SPARTAC, Swiss HIV Cohort Study, SALTO

CA-US HIV RNA: ACTG ATI Cohort Study

PD1 expression on CD4 and CD8 (prior to ART): SPARTAC

Yerly S, AIDS 2004; Williams JP, Elife 2014; Assoumou, AIDS 2015; Li JZ, AIDS 2016; Hurst, Nat Comms 2015

Conclusions

Multiple barriers to eliminating HIV including both HIV

persistence and impaired immunity

Agents that reverse latency need to be more potent,

more specific and induce death of the infected cell.

Alternative approaches may be needed to specifically

target different tissue reservoirs

Long term immune control also required to both boost T-

cell function and reduce inflammation. Future role of

immune checkpoint markers to be determined

New modalities needed to quantify the frequency and

location of infected cells and predict time of remission.

Acknowledgements

Doherty Institute, Uni Melb

Paul Cameron

Gabriela Khoury

Jenny Anderson

Hao Lu

Talia Mota

Thomas Rasmussen

Fiona Wightman

Ajantha Solomon

Surekha Tennakoon

Ashanti Dantanarayana

Renee Van der Sluis

Vanessa Evans

Michael Roche

Jori Symons

Julia Stout

Jennifer Audsley

The Alfred Hospital

Julian Elliott

James McMahon

Jennifer Hoy

Janine Roney

Michelle Bogliss

Burnet Institute

Melissa Churchill

Lachlan Gray

UCSF, San Francisco

Steven Deeks

Rebecca Hoh

Sulggi Lee

Wendy Hartogenesis

Peter Bacchetti

Rick Hecht

University of Montreal

Nicolas Chomont

Remi Fromentin

Leidos Laboratories, NCI

Jeff Lifson

Yerkes Primate Centre

Guido Silvestri

Merck

Richard Barnard

Australian HIV Cure

Community Partnership

Cipri Martinez

Brent Allen

Aaron Cogle