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Assay To Measure The Latent Reservoir Of Replication-Competent HIV-1 In Suppressed Patients Based On Ultra Deep Sequencing

Sook-Kyung LeeShuntai ZhouNancie Archin

David Margolis Ronald Swanstrom

 University of North Carolina

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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

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5 wells each containing 2 million resting T cells andone negative control well:

more cells, cultures can detect more measured events =greater accuracy

Ultra deep sequencing analysis ofculture 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

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1

Primer ID-based deep sequencing toquantitate virus number without limiting-dilution

IUPM Calculation – Correct For Unseen Duplicates In the Same Well1. Generate a phylogenetic tree using all distinct viral lineages observed from a

patient in all wells tested2. Count the number of wells containing the same viral lineage3. Determine the titer of all individual viral lineages based on Poisson distribution4. 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

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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

wellsC

1

C2

C3

C4

C5

C6

C7

C8

C9

C1

0

C1

10

20

40

60

80

100

120

% Distinct Viral Lineage in Chronic Patients

• Distinct viral sequences are often observed when cells are derived from chronic patients.

• Correct for duplicates with the Poisson distribution.

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IUPM-Ultra Deep Sequencing (UDS)Compared to IUPM-VOA

0 1 2 30

1

2

3

IUPM-VOA

IUP

M-U

DS

r=0.882P<0.0001

C1 C3 C5 C7 C9C11

0.00.51.01.52.02.53.03.5

Chronic Patients

IUP

M

IUPM-VOAIUPM-UDS

A strong correlation was observedbetween IUPM-VOA and IUPM-UDS

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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