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

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?. Resting T cells are latently infected early and persist Very low levels of ongoing replication occur.

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

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  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? • Resting T cells are latently infected early and persist • 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 Effective HAART should clear unintegrated HIV DNA T1/2 of Pre-integrated HIV DNA = 3 days PIC provirus HAART PIC PIC PIC provirus

  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. 4.5 y = -5.6132x + 20.825 3.5 2 R = 0.9764 Ln (proviruses) 2.5 1.5 0.5 3.1 3.2 3.3 3.4 3.5 3.6 3.7 Ln (Ct) 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 5.6 3.2

  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. 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 Acknowledgements

  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 6.5 1.5 1.4 Copies per cell 2.8 2.3 3.0 1.0

  24. An excess of unintegrated HIV DNA occurs in some patients on HAART 11* 2.0 3.2 1.6 1.8 1.8 5.9 5.6 0.01 0.001 Copies per cell 0.0001 0.00001 63 64 109 132 148 149 150 156 Patient number Total HIV (SST) Integrated HIV

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

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

  27. Assay of HIV integration

  28. 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.

  29. Validation of our assay

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