1. Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania

2. 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

3. 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.

4. provirus PIC provirus PIC HAART Effective HAART should clear unintegrated HIV DNA T 1/2 of Pre-integrated HIV DNA = 3 days

5. 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

6. Repetitive sampling of Integration standard

7. Validation of our assay

8. Validation of integration assay

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

10. An excess of unintegrated HIV DNA occurs off HAART

11. An excess of unintegrated HIV DNA occurs in Elite Suppressors

12. An excess of unintegrated HIV DNA occurs in some patients on HAART 11 3.2 5.6

13. One patient without an excess of unintegrated HIV DNA on HAART

14. Sporadic excess of unintegrated HIV DNA occur on HAART

15. Sporadic excesses of unintegrated HIV DNA on HAART

16. 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

17. 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

18. Reproducibility total/integrated HIV

19. Longitudinal samples suggest sporadic excess may occur

20. Sporadic excess of unintegrated HIV DNA occur on HAART

21. Sporadic blips of unintegrated HIV DNA on HAART

22. Overtime total HIV DNA approaches integrated HIV DNA on HAART Koelsch JID 2008

23. The level of unintegrated HIV DNA decreases with HAART 19 1.5 1.4 6.5 2.8 3.0 1.0 2.3 Copies per cell

25. An excess of unintegrated HIV DNA occurs in some patients on HAART 0.00001 0.0001 0.001 0.01 6364109132148149150156 Patient number Copies per cell Total HIV (SST)Integrated HIV 11*2.03.21.61.8 5.95.6

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

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

28. Assay of HIV integration

29. 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.

30. Validation of our assay