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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

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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)

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Defective viral genomes Ho et al. Cell 2013

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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 10-20 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

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TILDA in CD4 T cells from ART subjects The majority of cells with inducible virus are latently infected in ART subjects

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

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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

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

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

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

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

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

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

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Conclusions TILDA is: sensitive (1.4 cells/million) reproducible (coefficient of variation