1. Latent infection and cellular reservoirs
Where does HIV hide?
Latent infection and cellular reservoirs
Nicolas Chomont
Professionnal development workshop, July 21, Vienna

2. 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
START
STOP
HAART
Circulating virus
Limit of detection
Time
HIV hides in reservoir that are not sensitive to current therapies

3. 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)

4. How HIV persists? Persistence of latently infected cells
Ongoing viral replication
at low levels

5. How the HIV reservoir is established?Ag
Contraction
HIV
CCL19
Suha Saleh, Blood, 2007
Una O’Doherty, J Virol 2009

6. 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

7. How HIV Persists During Antiviral Therapy?
Total HIV DNA
Proviral HIV DNA
2-LTR circles
100000 ON
<50
+90
+22
+52 +3
+14
+71
+11
+24
10000
HIV DNA in 106 CD4 T cells
1000
100 10
HAART
Viral load
Weeks
OFF
3.107 -1
OFF
1.105 -1
OFF
3.104 -1
OFF
5.105
-0.5
During HAART, HIV persists as an integrated provirus

8. HIV Reservoir localization
Several cellular reservoirs have been described in vivo (CD4+, CD8+, CD3+CD4-CD8-, NK cells, Monocytes, Dendritic cells…)
3 major caveats:
Insufficient purity (results in false positive populations due to contamination by CD4 T cells)
Treatment duration too short (cellular reservoirs are different in viremic and HAART patients)
Sensitivity of the assay (results in false negative populations)

9. HIV Reservoir Localization
Strategy: cell sorting + quantification of HIV-1 DNA
CD8 PerCP Cy5.5
CD14 FITC
CD3 PE-Cy7
CD4 APC

10. HIV Reservoir Localization
Sorted CD4 T cells
Sorted CD8 T cells
Quantification of integrated HIV DNA by ultrasensitive real time PCR
Sorted DN T cells
Sorted Monocytes
CD8
CD14
CD3
CD4

11. HIV Integrated DNA in Sorted Cells
10000
1000
HIV DNA copies per 106 cells
100 10 1
CD4+
CD8+ DN
Monocytes
PBMC
HIV integrated DNA is rarely detected in non CD4 T cells from HAART patients

12. Contribution of cellular subsets to the HIV reservoir
100
Contribution to the pool of HIV infected cells 75 50 25
CD4+
CD8+ DN
Monocytes
CD4+ T cells constitute more than 95% of the HIV-1 reservoir
NON exhaustive approach: macrophages, NK, DC…?

13. HIV Reservoir Size

14. HIV Reservoir, decay? Time to eliminate: 105 cells: 54.8 years
Longitudinal measurements of the reservoir size indicate that this reservoir is extremely stable (half-life = months in subjects who have initiated HAART during chronic infection).
200000
400000
600000
800000
500
1000
Total number of reservoir cells
Linear scale
Time (months) 1 10
100
1000
10000
100000
200
400
600
800
Total number of reservoir cells
Log scale
Time (months)
Time to eliminate:
105 cells: 54.8 years
106 cells: 65.7 years
107 cells: 76.7 years

15. Factors impacting on the Reservoir Size2 4 1 10
100
1000
10000
% Ki67+ CD4 T cells
 = 0.44
p = 0.01
p <
10000
10000
1000
1000
Integrated HIV DNA copies per 106 CD4 T cells
Integrated HIV DNA copies per 106 CD4 T cells
Integrated HIV DNA copies per 106 CD4 T cells
100
100 10 10 1 1
< 1 year
> 1 year
< 1
> 1
Duration of exposure to HIV
CD4/CD8 ratio
Duration of exposure to HIV, CD4/CD8 ratio and Ki67 expression levels predict the HIV reservoir size

16. The CD4 compartment is heterogeneous
CD8 PerCP-Cy5.5
SSC
CD3 FITC
CD4 APC
Central memory cells: Long lived
SSC
Transitionnal memory cells: Slowly proliferate
CD45RA APC-Cy7
Naïve cells have not seen their cognate antigen
Effector memory cells: Immediate effector functions
CCR7 PE-Cy7
Terminally differentiated cells
CD27 PE

17. Contributions of CD4 T Cell Subsets to the HIV Reservoir
100 80
Contribution (%) to the HIV reservoir size 60 40 20 TN
TCM
TTM
TEM
TTD
CD45RA
CCR7
CD27 + - + - + - + -

18. CD4 Counts/Ki67 Levels and Reservoir Localization
100
 = 0.66
p = 0.004
 = -0.70
p = 0.002
 = 0.75
p =
% Ki67+ CD4 T cells 1 2 3 4 20 40 60 80
100 80
TCM contribution
%TCM cells among HIV+ cells 60 40 20
200
700
1200
CD4 count (cells/µl)
100
 = -0.64
p = 0.006 80
TTM contribution 60
%TTM+EM cells among HIV+ cells 40 20
200
700
1200
CD4 count (cells/µl)

19. Stability of the Reservoir in Patients with Proliferating TTM
10000 1
 = -0.88
p = 0.007
0.01
0.1 1 5 10
IL-7 (pg/ml)
 = -0.76
p = 0.037
1000
Integrated HIV DNA copies per 106 CD4 T cells
Slope decrease (% decrease/day)
0.1
100 10
0.01
400
800
1200 2 4 6
Days
Ki67+ TTM cells (%)
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.

20. 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

21. HAART HAART HAART HAART HAART HAART
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)
HAART
Cytokines, chemical compounds…
HAART
HAART
Uninfected cells
HAART
HAART
HAART
HIV-induced cell death
Interfering with the immunological mechanisms that contribute to HIV persistence:
T cell survival
Proliferation
Antibodies, cytokines, gene therapy, chemotherapy

22. 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

23. Acknowledgements The patients!! 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!!