2014 “Towards an HIV Cure” symposium
Melbourne
A novel assay that precisely measures the
size of the latent HIV reservoir reveals that
ART-naïve individuals harbour a large pool
of latently infected CD4+ T cells
Nicolas Chomont, PhD
Measuring the size of the reservoir
There is currently no gold standard method to measure the size of the latent
HIV reservoir (frequency of latently infected cells)
• The quantitative viral outgrowth assay measures replication-competent HIV but
may largely underestimate the size of the reservoir (Ho, Cell 2013)
• PCR based assays are reproducible, relatively easy to perform but may
overestimate the size of the reservoir (Eriksson, Plos Pathogens 2013)
There is a need to develop sensitive, reproducible and clinical trial
scalable methods to measure the size of the latent HIV reservoir
Defective viral genomes
Ho et al. Cell 2013
Principle of TILDA
TILDA: “Tat/Rev Induced Limiting Dilution Assay”
Ficoll gradient
centrifugation
10-20 mL
whole blood
Frequency of
cells with msHIV
RNA baseline
Negative
selection
PBMCs
CD4+
T cells
Nested RT-PCR
Distribute in
limiting dilutions for msHIV RNA
(24+40 cycles)
12h
PMA+ionomycin
Maximum likelihood
method
Frequency of
cells with
inducible msHIV
RNA
ART1
ART2
ART3
ART4
ART5
ART6
ART7
ART8
ART9
ART10
1000
100
10
1
Baseline
Induced
100
80
Induced
Baseline
60
40
20
0
AR
T1
AR
T2
AR
T3
AR
T4
AR
T5
AR
T6
AR
T7
AR
T8
AR
T9
AR
T1
0
Frequency of CD4+ T cells with
msRNA (cell/106 CD4+ T cells)
p =0.002
Contribution to the pool of cells
with inducible msRNA (%)
TILDA in CD4 T cells from ART subjects
The majority of cells with inducible virus are latently
infected in ART subjects
TILDA and other assays
x 27
x9
100000
x 13
x6
Frequency of HIV-infectedcells
(per 106 cells)
x 48
10000
1000
100
10
1
0.1
0
Viral outgrowth
Resting CD4
TILDA
Total CD4
1004
1033
1079
1126
1234
Tot. HIV DNA Tot. HIV DNA Int. HIV DNA Int. HIV DNA Tot. HIV DNA Residual viremia
PBMCs
Resting CD4
PBMCs
Resting CD4 GALT CD4
Plasma
(ddPCR)
(ddPCR)
(AluPCR)
(AluPCR)
(ddPCR)
2007
2021
2026
2056
2113
2147
2114
2177
2451
3068
3178
4001
2238
2248
2263
2264
2277
2286
2418
2420
2453
2454
TILDA gives a reservoir size in between Q-VOA and DNA
Adapted from Eriksson et al. 2013
TILDA and other assays
1
r = 0.381
p = 0.050
0.1
10
100
1000
100
10
1
r = 0.521
p = 0.056
10
100
100
r = 0.676
p = 0.002
1
1000
Total DNA in rectal CD4
10
100
r = 0.702
p = 0.002
100
10
1
0.10
10
100
1000
TILDA (cells expressing ms HIV RNA
per million CD4+ T cells)
100
1
10
100
1
0.10
r = -0.354
p = 0.070
0.01
1
10
100
1000
TILDA (cells expressing ms HIV RNA
per million CD4+ T cells)
1
1000
TILDA (cells expressing ms HIV RNA
per million CD4+ T cells)
r = 0.328
p = 0.095
0.01
1
r = 0.472
p = 0.076
10
Residual viremia
(copies/mL)
1000
1000
SCA
100
10000
10000
1000
Q-VOA
100000
Integrated DNA in resting CD4
TILDA (cells expressing ms HIV RNA
per million CD4+ T cells)
TILDA (cells expressing ms HIV RNA
per million CD4+ T cells)
TILDA (cells expressing ms HIV RNA
per million CD4+ T cells)
1000
10
0.1
1
1000
Viral outgrowth (IUPM)
1
10000
Integrated HIV DNA
(copies/106 resting CD4+ T cells)
10
Integrated DNA in PBMCs
10000
Integrated HIV DNA
(copies/106 PBMCs)
100
Rectal total HIV DNA
(copies/106 GALT CD4+ T cells)
Total HIV DNA
(copies/10 6 PBMCs)
1000
Total DNA in resting CD4
Total HIV DNA
(copies/106 resting CD4+ T cells)
Total DNA in PBMCs
10000
10
100
1000
TILDA (cells expressing ms HIV RNA
per million CD4+ T cells)
TILDA correlates with several assays measuring HIV persistence
ART in Acute and Chronic infection
TILDA
Total CD4
p=0.0089
Tot. HIV DNA
PBMCs
(ddPCR)
p=0.28
Tot. HIV DNA
Resting CD4
(ddPCR)
p=0.022
Int. HIV DNA
PBMCs
(AluPCR)
p=0.053
Int. HIV DNA
Resting CD4
(AluPCR)
p=0.18
Residual viremia
Plasma
10000
p=0.50
1000
1000
100
100
10
10
1
1
0.10
0.10
0.01
0.01
: ART started in acute infection
: ART started in chronic infection
TILDA distinguishes between subjects who have
started ART during acute and chronic infection
Plasma HIV-1 RNA
(copies/mL)
Frequency of HIV-infectedcells
(per 106 cells)
Viral outgrowth
Resting CD4
10000
p=0.069
TILDA in viremic subjects (no ART)
40
20
11
8
7
6
5
4
9
VI
R
VI
R
VI
R
VI
R
VI
R
VI
R
3
0
VI
R
Induced
60
2
Baseline
80
VI
R
1
Induced
Baseline
1
10
100
VI
R
100
VIR1
VIR2
VIR3
VIR4
VIR5
VIR6
VIR7
VIR8
VIR9
VIR10
VIR11
VIR12
VI
R
1000
Contribution to the pool of cells
with inducible msRNA (%)
Frequency of CD4+ T cells with
msRNA (cell/106 CD4+ T cells)
p =0.002
The majority of cells with inducible virus are latently
infected in ART naive subjects
TILDA in viremic subjects (no ART)
ART
VIR
Productively infected
CD4+ T cells
9.1%
90.9%
75.2%
24.8%
Latently infected
CD4+ T cells
75% of the cells with inducible HIV are latently infected
in untreated HIV infected subjects
Pre-integration latency?
Entry
Uncoating
Reverse
transcription
Viral
production
Integration
Viral
transcription
Post-integration
latency
Pre-integration
latency
Pre-integration latency?
TILDA
Post-integration
latency
PMA+ionomycin
RALTEGRAVIR
(4h)
Pre-integration
latency
+
PMA+ionomycin
+ RALTEGRAVIR
PMA+ionomycin
RALTEGRAVIR
(4h)
+
+
PMA+ionomycin
+ RALTEGRAVIR
-
Induced TILDA (cells expressing
ms HIV RNA per million CD4+ T cells)
2000
1500
1000
500
0
Induced TILDA (cells expressing
ms HIV RNA per million CD4+ T cells)
VIR1
2500
200
100
0
Induced TILDA (cells expressing
ms HIV RNA per million CD4+ T cells)
TILDA with raltegravir
VIR9
VIR11
300
1500
1000
500
0
Post-integration latency is already established in
untreated HIV-infected subjects
NS
PMA/iono
PMA/iono +RAL
Conclusions
 TILDA is:
• sensitive (1.4 cells/million)
• reproducible (coefficient of variation <0.2)
• fast (<2 days)
• relatively inexpensive ($300)
• easily transferable (basic culture set up + real time PCR)
• Clinical trial transferable (requires only 10mL of blood)
 The median frequency of “reservoir” cells measured by TILDA is 24 cells/million,
which is 48 times more than Q-VOA and 6 to 27 times less than PCR-based
assays
 In untreated disease, the frequency of latently infected cells largely exceeds the
frequency of productively infected cells suggesting that the majority of infected
cells are transcriptionally silent even in the absence of ART
 This provides a rationale for the use of shock and kill strategies at the time of
ART initiation
Shock and kill at ART initiation
ART
HIV
viral load
shock
HIV-specific CD8 T cells
Latent reservoir
ART
Acknowledgements
VGTI Florida
Francesco Procopio
Remi Fromentin
Deanna Kulpa
Amanda McNulty
Anne-Gaelle Blackwell
Lydie Trautmann
Rafick-Pierre Sekaly
Westmead Institute
Sarah Palmer
Karolinska Institutet
Susanne Eriksson
U19AI096109
R21AI113096
University of
Pennsylvania
Una O’Doherty
Merck and Co
Daria Hazuda
Mike Miller
Richard Barnard
Johns Hopkins
Robert Siliciano
Janet Siliciano
UCSF
Steven Deeks
Rick Hecht
UCSD
Doug Richman
Matt Strain
ARCHE 108687-54
The study
participants!