1. www.ias2015.org Assay To Measure The Latent Reservoir Of Replication-Competent HIV-1 In Suppressed Patients Based On Ultra Deep Sequencing Sook-Kyung Lee Shuntai Zhou Nancie Archin David Margolis Ronald Swanstrom University of North Carolina

2. www.ias2015.org New Assay Viral Outgrowth Assay (VOA) Detects replication competent virus: determines titer by end-point dilution Quantitative-Viral Outgrowth Assay: Current Gold Standard To Measure Latent Reservoir Of HIV-1

3. www.ias2015.org 5 wells each containing 2 million resting T cells and one negative control well: more cells, cultures can detect more measured events = greater accuracy Ultra deep sequencing analysis of culture supernatant of individual wells Culture for 7-14 days Count distinct viral lineages in each well Primer ID-Based Deep Sequencing Q-VOA 6-well plate: 5 cultures of 2 million resting T cells each

4. www.ias2015.org 1 Primer ID-based deep sequencing to quantitate virus number without limiting-dilution IUPM Calculation – Correct For Unseen Duplicates In the Same Well 1.Generate a phylogenetic tree using all distinct viral lineages observed from a patient in all wells tested 2.Count the number of wells containing the same viral lineage 3.Determine the titer of all individual viral lineages based on Poisson distribution 4.Add all the titers from the individual viral lineages Measuring the number of lineages per well, i.e. the number of different sequence variants 4 3 2

5. www.ias2015.org Hypothesis: Viral Outgrowth Lineages Induced From Different Cells Are Often Distinct In Chronic Patients Viral Outgrowth Assay (VOA) Viruses scored as p24 positive for the presence of HIV-1 from viral outgrowth assay Ultra deep sequencing analysis Phylogenetic tree to compare viral lineages derived from different wells Distinct viral sequences are often observed when cells are derived from chronic patients. Correct for duplicates with the Poisson distribution.

6. www.ias2015.org IUPM-Ultra Deep Sequencing (UDS) Compared to IUPM-VOA r=0.882 P<0.0001 IUPM-VOA IUPM-UDS A strong correlation was observed between IUPM-VOA and IUPM-UDS

7. www.ias2015.org o For all assays, accuracy is determined by number of events observed. o IUPM-UDS titers are strongly correlated with IUPM- VOA and allows the inclusion of all individual outgrowth events in determining the titer of the latent reservoir o IUPM can be under-estimated when the same viral sequences is induced from different cells in the same well. This problem is corrected by Poisson distribution. o IUPM can be over-estimated (i.e. artifactual lineages) due to recombinants produced during culture and PCR, and due to errors during cDNA synthesis and PCR. Inclusion of an abundance cut-off will remove most of this error but could lose some slow-growing viruses. Summary