Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University 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 PIC provirus PIC HAART Effective HAART should clear unintegrated HIV DNA T 1/2 of Pre-integrated HIV 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

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

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 HIV

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 Copies per cell

An excess of unintegrated HIV DNA occurs in some patients on HAART Patient number Copies per cell Total HIV (SST)Integrated HIV 11*

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