Epigenetic drug Gar1041 in combination with

antiretroviral therapy (ART) transiently reduces the proviral

DNA reservoir in SIVmac251-infected macaques

Andrea Savarino

Objectives

• To develop an antiretroviral treatment regimen in SIV infected macaques that could be used to test eradication strategies

• To increase the numbers and classes of effective antiviral drug for use in macaques that mirror treatments in human HIV cases

• To initiate preliminary studies to determine if virus eradication or disease alteration is possible

Virus Reservoirs Eradication

• Prevent any virus spread by intensified ART– Target multiple sites of viral inhibition

• Induce latently infected cells to replicate virus– Histone deacetylase inhibitors

• Eliminate productive/infected cells– Virus replication/cytotoxicity– Immune response– Passive therapies

Stimulation of HIV-1 LTR-controlled expression of GFP by MS-275 and buthionine sulfoximine (BSO) in a Jurkat cell clone (A1).

Savarino, et al., Retrovirology 2009.

Gar1041

ACH2 ctrl

0.6 μM GAR1041

M1

Reactive oxygen species (ROS)induction

NF-B nuclear translocation

Gar1041 72 h

Gar1041 12 h

Gar 1041 3 h

TNF- 72 h

Pos. control (TNF- 1.5

h)

p24 induction

Gar1041

Gar1041

0 103 104 105

<FITC-A>: CD27

0

50K

100K

150K

200K

250K

SS

C-A

18.2

0 103 104 105

<FITC-A>: CD27

0

50K

100K

150K

200K

250K

SS

C-A

28.9

0 103 104 105

<FITC-A>: CD27

0

50K

100K

150K

200K

250K

SS

C-A

94.9

0 103 104 105

<FITC-A>: CD27

0

50K

100K

150K

200K

250K

SS

C-A

94.2

0 103 104 105

<FITC-A>: CD27

0

50K

100K

150K

200K

250K

SS

C-A

5.13

0 103 104 105

<FITC-A>: CD27

0

50K

100K

150K

200K

250K

SS

C-A

29.8

0 103 104 105

<FITC-A>: CD27

0

50K

100K

150K

200K

250K

SS

C-A

4.7

0 103 104 105

<FITC-A>: CD27

0

50K

100K

150K

200K

250K

SS

C-A

10.6

CD27 Downregulation

TN TCM TTM TEM

Mock

50nM Gar1041

Response of SIVmac251-infected macaques to raltegravir

0 10 20 30 40 50101

102

103

104

105

106

107

P044P249

P252P255

M970

M974

Days

Vir

al lo

ad

(RN

A c

op

ies

ml-1

)

RAL RAL + PMPA + FTC

RAL- 100mg BID, oral

PMPA - 20mg/kg, SQ

FTC – 50mg/kg, SQ

-200 -150 -100 -50 0 500

1000

2000

P044P249

P252P255

M970

M974

Days

CD

4+ T

-lym

ph

ocy

tes

l-1

RAL RAL + PMPA + FTC

0 20 40 600

20

40

60

80

100

P044P249P252P255M970M974

nonhuman primates

Days of treatment

DN

A c

op

ies/

5*1

05 c

ells

Lewis M. Norelli S., et al., Retrovirology 2010.

Treatment of monkeys with ART plus Gar1041

0 20 40 6010

100

1000P044P249P252P255M970M974

Days of treatment

RN

A C

op

ies/

ml

0 20 40 601

10

100

1000P044P249P252P255M970M974

******

***

* ***

pro

vir

al D

NA

co

pie

s

*P < 0.05 **P < 0.01***P < 0.001

A

0 300

20

40

60

80

100

*

Days of treatmentwith ART + auranofin

% o

f C

D4

+T

CM

lym

ph

oc

yte

s

B

0 300

20

40

60

80

*

Days of treatmentwith ART + auranofin

% o

f C

D4+

TE

M ly

mp

ho

cyt

esC

0 300

1000

2000

3000

Days of treatmentwith ART + auranofin

tota

l CD

4+ T

-ly

mp

ho

cy

tes

/l o

f b

loo

d

P < 0.05

P < 0.05

SIVmac251 inhibition in CEMx174cells

0

20

40

60

80

100

0.1 1 10 100 1000

EC50 = 6.6 nMP = 0.02

DRV conc. [nM]

% in

hib

itio

n

Darunavir (DRV) complexed with HIV-2 protease DRV complexed with SIVmac251 protease

Barreca ML, Norelli S, and Savarino A,unpublished

Kovalevsky et al., 2008

B

0 250

1000

2000

3000

*

Days of ART intensification

CD

4+ T

-Cel

ls/

l

A

0 250

200

400

600

800

Days of ART intensification

RN

A c

op

ies/

ml o

f p

lasm

aAddition of Ritonavir-boosted Darunavir to the ART/Gar1041 regimen

*P < 0.05

iART alone

0 20 40 60 80 1000

102030405060708090

100110

44234416Mean

Days of ART intensificationp

rov

ira

l DN

A c

op

ies

/ 5*1

05 c

ells

iART + Gar1041

0 50 100 1500

10

20

30

40

50

60 M974M970P255P252P249P044MEAN

Days of ART intensification

pro

vira

l DN

A c

op

ies

/ 5*

105 c

ells

iART/Gar1041 (average trend over time: P = 0.0207; t-test for regression). iART alone (average trend over time: P = 0.8878; t-test for regression).

Proviral DNA in PBMC from SIV+ Macaques Following Addition of Ritonavir-boosted Darunavir ART/Gar1041 Regimen.

Stimulation of viral replication by histone deacetylase inhibitor, SAHA in SIVmac251-infected monkeys treated with intensified ART (iART) alone but not in

monkeys SIVmac251-infected monkeys treated with iART and Gar1041

-20 -10 10 20 30 40 50 60 70 8010

100

1000

10000

100000

100000043884488

0

***

**

******

****

******

****

**

**

* *

P044P249P252P255M970M972

therapysuspesion

Monkeys:

Days from SAHA start

Vir

al l

oa

d(R

NA

co

pie

s m

l-1) iART alone

iART plus Gar1041

*P < 0.05 **P < 0.01

0 2 4 6 8 100

25

50

75

100iART plus auranofiniART alone

A

weeks following treatment interruption

per

cen

tag

e o

f an

imal

s sh

ow

ing

un

det

ecta

ble

vir

al lo

ads

0 2 4 6 8 100

25

50

75

1004-SAHA cycles3 SAHA cycles

B

weeks following treatment interruption

per

cen

tag

e o

f an

imal

s sh

ow

ing

un

det

ecta

ble

vir

al lo

ads

Re-appearance of viral loads following treatment suspension

*P < 0.05; Gehan-Breslow-Wilcoxon test for survival

*

*

Intensified ART aloneIntensified ART plus Gar1041

3 SAHA cycles2 SAHA cycles

*P < 0.05; paired Student’s t-test

Drug free remission… eventually?

iART alone

iART plus Gar1041

Drug free remission… eventually?

iART alone

iART plus Gar1041

Conclusions

Our findings suggest that Gar1041 impacts the proviral DNA reservoir in SIVmac251-infected macaques under treatment with ART, likely acting by an entirely novel mechanism (differentiation of long-lived or proliferation-competent memory T cells into short-lived effector-like phenotypes).

They also highlight the need for ART intensification when adopting drugs capable of inducing HIV-1 replication.

Preliminary studies using oxidative stress inducing agents indicate the utility of the simian model to assess in-vivo the effectiveness of treatments that may stimulate the removal of latently infected cells

AcknowledgmentsBioqualMatt CollinsJake Yalley-Ogunro University of Rome “Tor Vergata” Jack Greenhouse Enrico GaraciWendy Wagner

ICGEBIstituto Superiore di Sanità, Rome Marina LusicSandro NorelliBarbara Chirullo University of Rome, La

SapienzaMarco Sgarbanti Rossella SgarbantiAndrea Savarino Anna Teresa Palamara