T cell survival (TCM)

Report
Where does HIV hide?
Latent infection and cellular reservoirs
Nicolas Chomont
Professionnal development workshop, July 21, Vienna
Current Anti-HIV Drugs do not Eradicate HIV
HIV infection is characterized
by high levels of circulating
viruses in the blood
Antiretroviral drugs (HAART) are
capable of suppressing HIV, even to
undetectable levels
However, the virus
rebounds after
cessation of therapy
STOP
START
Circulating virus
HAART
Limit of detection
Time
HIV hides in reservoir that are not sensitive to current therapies
How HIV persists?
1995:
Resting CD4 T cells harbouring integrated provirus are present in
HIV-infected individuals (TW Chun, Nat. Med).
1997:
These cells persist in patients receiving suppressive HAART
(D Finzi, Science ; TW Chun, PNAS).
1999:
The half life of this reservoir is extremely long (D Finzi Nat. Med) but can
be reduced by initiating HAART in acute infection (L Zhang, NEJM).
2005:
Ongoing viral replication occurs in subjects on suppressive HAART
and contributes to HIV persistence (TW Chun, JCI ; N Tobin, J Virol).
Now:
HAART intensification studies
(J Dinoso, PNAS ; D McMahon, CID; M Buzon, Nat Med)
How HIV persists?
Persistence of
latently infected cells
Ongoing viral replication
at low levels
How the HIV reservoir is established?
HIV
Contraction
Ag
HIV
CCL19
Suha Saleh, Blood, 2007
Una O’Doherty, J Virol 2009
Where HIV Persists During Antiviral Therapy?
• At the anatomical level: Potential “hiding places” for HIV:
• Brain
• Lymph nodes
• Peripheral blood
• Gut
• Bone marrow
• At the cellular level: A small pool of cells harbor
viral DNA integrated within the genome of the
host. The frequency of these “reservoir cells” is
very low (less than 1 in a million)
HIV can hide “dormant” for the lifetime of the cell
How HIV Persists During Antiviral Therapy?
Total HIV DNA
Proviral HIV DNA
2-LTR circles
100000
10000
1000
100
10
HAART
Viral load
Weeks
OFF ON
3.107 <50
-1
+52
ON
<50
+90
OFF ON
1.105 <50
-1
+3
ON
<50
+22
OFF ON
3.104 <50
-1
+14
ON
<50
+71
OFF ON
5.105 <50
-0.5 +11
ON
<50
+24
During HAART, HIV persists as an integrated provirus
HIV Reservoir localization
Several cellular reservoirs have been described in vivo (CD4+, CD8+,
CD3+CD4-CD8-, NK cells, Monocytes, Dendritic cells…)
3 major caveats:
1. Insufficient purity (results in false positive populations due to contamination
by CD4 T cells)
2. Treatment duration too short (cellular reservoirs are different in viremic and
HAART patients)
3. Sensitivity of the assay (results in false negative populations)
HIV Reservoir Localization
CD14 FITC
CD8 PerCP Cy5.5
Strategy: cell sorting + quantification of HIV-1 DNA
CD3 PE-Cy7
CD4 APC
HIV Reservoir Localization
Sorted CD4 T cells
Sorted CD8 T cells
Quantification of integrated
HIV DNA by ultrasensitive
real time PCR
CD14
Sorted Monocytes
CD8
Sorted DN T cells
CD3
CD4
HIV Integrated DNA in Sorted Cells
HIV DNA copies per 106 cells
10000
1000
100
10
1
CD4+
CD8+
DN
Monocytes
PBMC
HIV integrated DNA is rarely detected in non CD4 T cells from HAART patients
Contribution to the pool of
HIV infected cells
Contribution of cellular subsets to the HIV reservoir
100
75
50
25
0
CD4+
CD8+
DN
Monocytes
CD4+ T cells constitute more than 95% of the HIV-1 reservoir
NON exhaustive approach: macrophages, NK, DC…?
HIV Reservoir Size
HIV Reservoir, decay?
Longitudinal measurements of the reservoir size indicate that this reservoir is
extremely stable (half-life = 39-44 months in subjects who have initiated HAART
during chronic infection).
Linear scale
Log scale
1200000
1000000
1000000
100000
800000
10000
600000
1000
400000
100
200000
10
0
1
0
500
1000
0
Time (months)
200
400
600
800
Time (months)
Time to eliminate:
105 cells: 54.8 years
106 cells: 65.7 years
107 cells: 76.7 years
1000
1000
100
10
1
< 1 year > 1 year
Duration of exposure to HIV
10000
p < 0.0001
1000
100
10
1
<1
>1
CD4/CD8 ratio
Integrated HIV DNA copies
per 106 CD4 T cells
10000
p < 0.0001
Integrated HIV DNA copies
per 106 CD4 T cells
Integrated HIV DNA copies
per 106 CD4 T cells
Factors impacting on the Reservoir Size
10000
1000
100
10
 = 0.44
p = 0.01
1
0
2
% Ki67+ CD4 T cells
Duration of exposure to HIV, CD4/CD8 ratio and Ki67 expression
levels predict the HIV reservoir size
4
SSC
CD8 PerCP-Cy5.5
The CD4 compartment is heterogeneous
CD3 FITC
CD4 APC
SSC
Central memory
cells: Long lived
Transitionnal memory
cells: Slowly proliferate
CCR7 PE-Cy7
Effector memory
cells: Immediate
effector functions
CD45RA APC-Cy7
CD27 PE
Naïve cells have
not seen their
cognate antigen
Terminally
differentiated cells
Contribution (%) to the HIV reservoir size
Contributions of CD4 T Cell Subsets to the HIV Reservoir
100
80
60
40
20
0
TN
CD45RA
CCR7
CD27
+
+
+
TCM
TTM
TEM
TTD
+
+
+
-
+
-
TTM contribution
%TTM+EM cells among HIV+ cells
TCM contribution
%TCM cells among HIV+ cells
CD4 Counts/Ki67 Levels and Reservoir Localization
100
80
 = 0.66
p = 0.004
 = -0.70
p = 0.002
100
80
60
60
40
40
20
20
0
200
100
80
700
1200
CD4 count (cells/µl)
 = -0.64
p = 0.006
0
0
 = 0.75
p = 0.0006
100
80
60
60
40
40
20
20
0
200
1
2
3
4
% Ki67+ CD4 T cells
0
700
1200
CD4 count (cells/µl)
0
1
2
3
4
% Ki67+ CD4 T cells
Integrated HIV DNA copies
per 106 CD4 T cells
Stability of the Reservoir in Patients with Proliferating TTM
10000
1
 = -0.88
p = 0.007
1000
1
 = -0.76
p = 0.037
0.1
0.1
100
10
0
400
Days
800
1200
0.01
0.01
0
2
4
6
Ki67+ TTM cells (%)
0
5
IL-7 (pg/ml)
Stability of the HIV reservoir size in patients with proliferating TTM and high IL-7 levels
=> Homeostatic proliferation of reservoir cells contribute to HIV persistence.
10
How HIV persists during antiviral therapy?
At least three mechanisms could be implicated:
• Ongoing viral replication at low levels and replenishment of the pool of infected
cells (particularly in privileged anatomical reservoirs such as the central nervous
system and the gut).
• T cell survival (TCM): Reservoir cells are memory T cells. These cells, which are
generated after infection or vaccination, keep the memory of the immune system for
decades.
• Proliferation (TTM): Reservoir cells, like other memory T cells, divide very slowly to
maintain the memory of the immune system (A. Bosque and V. Planelles)
Viral replication
T cell survival
Proliferation
Can we reduce the HIV reservoir size?
Possible strategies to eliminate/control/reduce the reservoir:
• Start HAART during acute infection (Hocqueloux, AIDS 2010)
• Intensification of HAART
• Reactivation of HIV replication from its latent reservoir (D. Margolis, A. Savarino)
Cytokines, chemical
compounds…
Uninfected cells
HIV-induced cell death
• Interfering with the immunological mechanisms that contribute to HIV persistence:
T cell survival
Antibodies, cytokines, gene
therapy, chemotherapy
Proliferation
Conclusion
• In individuals on suppressive HAART, HIV persists primarily in CD4 T cells.
• The HIV reservoir is stable and 70 years of continuous suppressive HAART may be
necessary to eradicate HIV.
• Early HAART initiation limits the size of the viral reservoir.
• Within the CD4 compartment, TCM (long lived) and TTM (slowly proliferating)
constitute the 2 majors viral reservoirs.
• At least 3 mechanisms contribute to HIV persistence:
- Ongoing viral replication (Gut, CNS etc…)
- T cell survival
- Homeostatic proliferation
Their relative contributions are unknown but are likely to differ between individuals
Acknowledgements
VGTI Florida
Université de Montréal, CHUM
INSERM U743
Sandrina Da Fonseca
Claire Vandergeeten
Mohamed El Far
Petronela Ancuta
Lydie Trautmann
Francesco Procopio
Bader Yassine-Diab
Genevieve Boucher
Elias Haddad
Rafick-Pierre Sékaly
Royal Victoria Hospital, Mc Gill University
Jean-Pierre Routy
Mohamed-Rachid Boulassel
Georges Ghattas
VRC, NIAID, NIH
Danny Douek
Jason Brenchley
Brenna Hill
The patients!!

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