2014 “Towards an HIV Cure” symposium Melbourne A novel assay that precisely measures the size of the latent HIV reservoir reveals that ART- nave individuals harbour a large pool of latently infected CD4+ T cells Nicolas Chomont, PhD
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Slide 1
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