Latent infection and cellular reservoirs 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 START STOP HAART Circulating virus 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? Ag Contraction 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 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

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)

HIV Reservoir Localization Strategy: cell sorting + quantification of HIV-1 DNA CD8 PerCP Cy5.5 CD14 FITC 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 Sorted DN T cells Sorted Monocytes CD8 CD14 CD3 CD4

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

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…?

HIV Reservoir Size

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 = 39-44 months in subjects who have initiated HAART during chronic infection). 200000 400000 600000 800000 1000000 1200000 500 1000 Total number of reservoir cells Linear scale Time (months) 1 10 100 1000 10000 100000 1000000 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

Factors impacting on the Reservoir Size 2 4 1 10 100 1000 10000 % Ki67+ CD4 T cells  = 0.44 p = 0.01 p < 0.0001 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

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

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 + - + - + - + -

CD4 Counts/Ki67 Levels and Reservoir Localization 100  = 0.66 p = 0.004  = -0.70 p = 0.002  = 0.75 p = 0.0006 % 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)

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.

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

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

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 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!!