Ex vivo modeling of HIV persistence in successfully treated subjects ...

“Towards an HIV Cure”
Pre-Conference Symposium
20 & 21 July 2012
Ex vivo modeling of HIV persistence in successfully
treated subjects: A powerful tool to evaluate novel
therapeutic eradication strategies
Rémi Fromentin
Vaccine and Gene Therapy Institute-Florida,
Port St Lucie, FL, USA

Evaluative tools for an HIV cure
Crucial need to develop powerful tools:
• To test novel therapeutic strategies
• To predict their clinical outcome
• To evaluate their capacity to eliminate
latently infected cells

• Current methods to study HIV latency:
- Cell lines
- Primary human cells (Bosque, Blood 2009; Marini, JI 2008; Tyagi, JV 2010;
Swiggard, JV 2005; Saleh, Blood 2007; Yang, JCI 2009)
- Cells from virally suppressed subjects (Reuse, PLoS One 2009;
Blazkova, JID 2012)
Current tools
• Current assays to measure HIV persistence:
- HIV DNA
- Cell-associated HIV RNA
- Co-culture assay (IUPM)
- Single Copy Assay (SCA)

HIV Persistence Detection Assay
“HPDA”
• 5.10 6 CD4 T cells from virally suppressed subjects
cultured in 1mL of RPMI 10% FBS + AZT, RAL, EFV
• Harvest half the medium at day 3 and 6
• vRNA RT-qPCR on ultracentifuged supernatants
ARVs
CD4 T cells from virally
suppressed HIV infected subjects
Quantification by RT-qPCR with
a sensitivity of 1 HIV RNA copy

HPDA: Measuring the HIV reservoir size
Viral production
(HIV RNA per 10 6 CD4 T cells)
10000
1000
100
10
1
0.1
NS
CD3/CD28
The HIV Persistence Detection Assay (HPDA) is sensitive and presents a
wide dynamic range of detection.

HPDA: Measuring the HIV reservoir size
Viral production
(HIV RNA per 10 6 CD4 T cells)
1000
100
10
1
0.1
p=0.05
0 250 500 750 1000 1250
Integrated HIV DNA
per 10 6 CD4 T cells
Viral production
(HIV RNA per 10 6 CD4 T cells)
10000
1000
100
10
p

HPDA: Measuring the HIV reservoir size
in tissues
Viral production
(HIV RNA per 10 6 CD4 T cells)
100000
10000
1000
100
10
1
A
B
C
Virally suppressed subjects
Peripheral Blood
Lymph Node
The HPDA allows the measurement of the HIV reservoir size in tissues
from virally suppressed subjects.

HPDA: Measuring the HIV reservoir size
Assay
qPCR
HIV reservoir measured by
Number of events
Integrated HIV DNA
Pros and cons
+ : Easy, fast, sensitive
- : The majority of integrated
DNA is defective
HIV Persistence Detection Assay
ARV
Production competent virus
+ : Relatively easy and fast
+ : Useful to test reactivation
agents
+ : Measure the frequency of
cells directly targeted by
reactivation strategies
co-culture assay
Allogenic cells
Replication competent virus
- : Laborious and costly
+ : Measure the frequency of cells
able to produce infectious particles
responsible for viral rebound at
ART interruption.
+ : Readout for sterilized cure
Cost, labor

HPDA: Identifying reactivation strategies
Reactivation
(% relative to CD3/CD28)
40
30
20
10
0
n=3
Known anti-latency agents:
PKC activators : prostratin, bryostatin
HDAC inhibitors : SAHA, VPA
The HPDA allows the evaluation of the ability of anti-latency agents to
reactivate virus in latently infected cells from virally suppressed subjects.

HPDA: Identifying reactivation strategies
Reactivation
(% relative to CD3/CD28)
15
10
5
0
Subject A
10.0
7.5
5.0
2.5
0.0
Subject B
100
75
50
25
0
Subject C
The response of latently infected cells to anti-latency agents is highly
variable between virally suppressed subjects.

HPDA: Identifying novel antiviral compounds
In addition to viral latency, residual levels of HIV production may contribute to HIV
persistence.
didehydro-Cortistatin A (dCA) = Tat inhibitor

HPDA: Identifying novel antiviral compounds
Viral production
(HIV RNA copies)
10000
1000
100
10
DMSO
dCA 100nM
1
A B C D
Virally suppressed subjects
dCA inhibits spontaneous viral release from persistently infected cells from virally
suppressed subjects.
HPDA allows the evaluation of antiviral compounds aimed at affecting ongoing viral
production that persists under HAART.

Conclusions
• Therapeutic strategies aimed at reducing the size of the HIV
reservoir are likely to differ between subjects.
• HPDA allows the identification of personalized eradication
strategies.

Conclusions
The HIV Persistence Detection Assay:
• Is standardized and easy to perform.
• Uses a relatively low number of cells.
• Directly measures the reactivable HIV reservoir which is the
target of current curative strategies.
HPDA is a simple, accurate and clinical trial scalable assay to
measure HIV persistence.

Acknowledgement
Volunteers
Claire Vandergeeten
Mariam Lawani
Sandrina Dafonseca
Rafick-Pierre Sekaly
Nicolas Chomont
Supported by
Susana T Valente
Mike D Miller
Daria Hazuda