2014 “Towards an HIV Cure” symposium Melbourne A novel assay that precisely measures the size of the latent HIV reservoir reveals that ART-nai ̈ ve individuals harbour a large pool of latently infected CD4+ T cells Nicolas Chomont, PhD

Measuring the size of the reservoir There is a need to develop sensitive, reproducible and clinical trial scalable methods to measure the size of the latent HIV 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)

Defective viral genomes Ho et al. Cell 2013

Principle of TILDA TILDA: “Tat/Rev Induced Limiting Dilution Assay” Nested RT-PCR for msHIV RNA (24+40 cycles) Maximum likelihood method Frequency of cells with inducible msHIV RNA mL whole blood PBMCs Ficoll gradient centrifugation CD4 + T cells Negative selection 12h PMA+ionomycin Distribute in limiting dilutions Frequency of cells with msHIV RNA baseline

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 TILDA gives a reservoir size in between Q-VOA and DNA Adapted from Eriksson et al. 2013

TILDA and other assays Total DNA in PBMCsTotal DNA in resting CD4Integrated DNA in resting CD4Integrated DNA in PBMCs Total DNA in rectal CD4Q-VOASCA TILDA correlates with several assays measuring HIV persistence

ART in Acute and Chronic infection TILDA distinguishes between subjects who have started ART during acute and chronic infection

TILDA in viremic subjects (no ART) The majority of cells with inducible virus are latently infected in ART naive subjects

TILDA in viremic subjects (no ART) 75% of the cells with inducible HIV are latently infected in untreated HIV infected subjects ARTVIR

Pre-integration latency? Pre-integration latency Post-integration latency Entry Uncoating Reverse transcription Integration Viral transcription Viral production

Pre-integration latency? Pre-integration latency Post-integration latency PMA+ionomycin TILDA + + RALTEGRAVIR (4h) PMA+ionomycin + RALTEGRAVIR RALTEGRAVIR (4h) PMA+ionomycin + RALTEGRAVIR + -

TILDA with raltegravir VIR1VIR9VIR11 Post-integration latency is already established in untreated HIV-infected subjects

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 shock HIV-specific CD8 T cells Latent reservoir HIV viral load ART

Acknowledgements VGTI Florida Francesco Procopio Remi Fromentin Deanna Kulpa Amanda McNulty Anne-Gaelle Blackwell Lydie Trautmann Rafick-Pierre Sekaly Merck and Co Daria Hazuda Mike Miller Richard Barnard UCSF Steven Deeks Rick Hecht Westmead Institute Sarah Palmer Karolinska Institutet Susanne Eriksson University of Pennsylvania Una O’Doherty Johns Hopkins Robert Siliciano Janet Siliciano UCSD Doug Richman Matt Strain The study participants! U19AI096109R21AI ARCHE