Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on

HAART

Una O’DohertyUniversity of Pennsylvania

How are reservoirs of HIV maintained in patients on HAART?

1. Resting T cells are latently infected early and persist

2. Very low levels of ongoing replication occur

Unclear if ongoing replication may occur on HAART

• Against:– viral evolution studies – Intensification studies

• However: – Low levels of ongoing replication– Sporadic ongoing replication– Only occur in a subset of HAART patients– Evidence in:

• HIV DNA in aCD4 (Chun et al 2005) • 2-LTR with Raltegravir (Buzon et al 2010) • Half-life of unintegrated HIV DNA (Koelsch et al 2008 and Chomont et

al 2009)

• Thus, we need a sensitive method to detect viral replication.

provirus

provirus

PIC PIC

PIC

PIC

PIC

provirus

provirus

PIC

HAART

Effective HAART should clear unintegrated HIV DNA

T1/2 of Pre-integratedHIV DNA = 3 days

Generation of the Integration Standard (IS) cell line

• The standard contains the following:– Only integrated HIV DNA– Multipe integration sites to reflect the high level of diversity of

integration site selection during natural infection

Repetitive sampling of Integration standard

y = -5.6132x + 20.825R2 = 0.9764

0.5

1.5

2.5

3.5

4.5

3.1 3.2 3.3 3.4 3.5 3.6 3.7Ln (Ct)

Ln (p

rovi

ruse

s)

Validation of our assay

Validation of integration assay

Confidence interval: ratio of total HIV DNA/integrated with our integration standard

An excess of unintegrated HIV DNA occurs off HAART

An excess of unintegrated HIV DNA occurs in Elite Suppressors

An excess of unintegrated HIV DNA occurs in some patients on HAART

11

3.2

0.00001

0.0001

0.001

0.01

1 2 3 4 5 6 7

Cop

ies

per c

ell

Total HIV DNAIntegrated HIV DNA

5.6

One patient without an excess of unintegrated HIV DNA on HAART

Sporadic excess of unintegrated HIV DNA occur on HAART

Sporadic excesses of unintegrated HIV DNA on HAART

Conclusions

• A fraction of patients on HAART have a clear excess of unintegrated HIV DNA

• Longitudinal study suggests excess may occur sporadically

• An excess of unintegrated DNA may provide a surrogate marker for ongoing replication

Acknowledgements• My lab

– Angela Mexas– Erin Graf– Jianqing Yu– Luis Agosto– Matthew Pace

• Funding– Merck, NIH

• NIH NIAID– Stephen Migueles– Mark Connors– Tae-Wook Chun

• Penn– Robert Gross– Ian Frank– Bruce Levine– Avinash Bhandoola

• Merck– Daria Hazuda

Reproducibility total/integrated HIVRU5/cell Provirus/cell Total/Integrated0.00052 0.00018 1.40.00045 0.00016 1.40.00044 0.00014 1.60.00063 0.00011 2.90.00044 0.00012 1.90.00044 0.00012 1.9

AverageStd devRange

1.830.55

1.4 - 2.9

Total (RU5/2)/ integrated

Longitudinal samples suggest sporadic excess may occur

Sporadic excess of unintegrated HIV DNA occur on HAART

Sporadic blips of unintegrated HIV DNA on HAART

Overtime total HIV DNA approaches integrated HIV DNA on HAART

Koelsch JID 2008

The level of unintegrated HIV DNA decreases with HAART

19

1.5 1.4

6.5

2.8

3.01.0

2.3

Cop

ies

per c

ell

An excess of unintegrated HIV DNA occurs in some patients on HAART

0.00001

0.0001

0.001

0.01

63 64 109 132 148 149 150 156

Patient number

Cop

ies

per c

ell

Total HIV (SST) Integrated HIV

11* 2.0 3.2 1.6 1.8 1.8 5.9 5.6

The half-life of unintegrated HIV DNA is ~ 3 days in rCD4+T cells treated

An excess of unintegrated HIV DNA occurs in some patients on HAART

Assay of HIV integration

Unclear if ongoing replication may occur on HAART

• Against– viral evolution studies – Intensification studies

• However: – Low levels of ongoing replication may occur without

detectable evolution– Replication may occur sporadically– Replication may occur in a subset of patients on HAART– Buzon et al 2010, Koelsch et al 2008 and Chomont et al 2009

• Thus, we need a sensitive method to detect viral replication.

Validation of our assay