1. Interactive Workshop “HIV Cure 101”: Challenges in identifying and targeting the HIV reservoir Sarah Palmer Centre for Virus Research Westmead Millennium Institute for Medical Research University of Sydney

2. Definitions Residual Viremia: Persistent HIV RNA measured in plasma at levels below the limit of detection of the standard clinical assay (20-50 copies/ml). Viral Reservoir: A viral reservoir is an anatomical site or cell type in which a replication-competent form of HIV persists, accumulating with more stable properties than the circulating pool of actively replicating virus. This HIV can persist even during effective therapy.

3. 0 200 400 600 800 Time (days) 10 7 10 7 10 6 10 6 10 5 10 5 10 4 10 4 10 3 10 3 10 2 10 2 10 1 10 1 10 0 10 0 10 -1 10 -1 0 200 400 600 800 Time (days) 10 7 10 7 10 6 10 6 10 5 10 5 10 4 10 4 10 3 10 3 10 2 10 2 10 1 10 1 10 0 10 0 10 -1 10 -1 Plasma HIV-1 RNA (copies/ml) 22 ± 6 c/ml 4 ± 2 c/ml Viremia Persists after Suppression by Antiretroviral Therapy Start Therapy (d4T/3TC/efavirenz) bDNA bDNA <75 copies/ml Single-Copy Assay Single-Copy Assay < 1 copy/ml Palmer et al. JCM 2003 Maldarelli et al. PLoS Path 2007

4. 0.32 1 3.2 10 32 100 Biphasic decline in persistent viremia over 7 years of treatment 11.6 copies/ml half-life=39 weeks 1.5 copies/ml 720 study assay limit 720 study Palmer et al. PNAS 2008 half-life= ∞

5. Persistent HIV Infection The IAS Scientific Working Group on HIV Cure: Nature Reviews Immunology 2012 Palmer et al. JIM 2011

6. www.aids2014.org Persistent HIV Infection

7. www.aids2014.org Measuring Persistent HIV Where to Measure Persistent Virus?  Peripheral Blood Plasma Cells: RNA versus DNA  Tissue Compartments T cells Other cell types RNA versus DNA  Role of Replication Defective HIV CNS/CSF

8. www.aids2014.org Measuring Persistent HIV Culture assay : IUPM Lewin & Rouzioux, AIDS 2011 Rouzioux & Richman, 2012

9. www.aids2014.org Measuring Persistent HIV Infection MeasurementAdvantagesDisadvantages HIV RNA in PlasmaRelatively inexpensiveDifficult to separate reservoir expression vs HIV replication, some positive samples undetectable, may not be representative of intracellular HIV RNA and DNA levels Infectious Virus: estimates the number of infectious units of HIV per million mononuclear cells (IUPM) Only direct measurement of replication competent virus or number of proviruses capable of productive infection Requires large quantities of cells, $$$, large error, often impossible to detect changes in reservoir size Total HIV DNA (Peripheral Blood or Tissue Compartments) Inexpensive, easyUnintegrated HIV DNA contributes to signal unless patients are on HAART for 1-3 years. A lot of virus is defective. Integrated HIV DNA (Peripheral Blood or Tissue Compartments) excludes unintegrated HIV DNA, less error than IUPM Requires at least a million cells, complex assay, defective provirus

10. www.aids2014.org Persistent HIV Infection in Tissue CNS/CSF

11. www.aids2014.org Persistent HIV Infection in Tissues Svicher et al. Curr HIV/AIDs Rep. 2014

12. www.aids2014.org Looking Ahead Does a “cure” require the total absence of HIV RNA and DNA? If not, then new more sensitive assays will be needed to differentiate between replication competent and non-replicating virus. Must all potential reservoirs be analyzed? If not, are we confident that certain reservoirs are determinative of cure/remission? With advances in curative strategies, will our current sensitive assays provide sufficient confidence to stop HIV therapy in patients showing a near absence of HIV RNA and DNA? Looking ahead, to determine the effectiveness of curative strategies, our field will need to develop a more standardized assay system which is sensitive, efficient, less costly, and adoptable in local settings.