Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty...
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Transcript of Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty...
![Page 1: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/1.jpg)
Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on
HAART
Una O’Doherty
University of Pennsylvania
![Page 2: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/2.jpg)
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
![Page 3: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/3.jpg)
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.
![Page 4: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/4.jpg)
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
![Page 5: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/5.jpg)
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
![Page 6: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/6.jpg)
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 (
prov
iruse
s)
![Page 7: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/7.jpg)
Validation of our assay
![Page 8: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/8.jpg)
Validation of integration assay
![Page 9: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/9.jpg)
Confidence interval: ratio of total HIV DNA/integrated with our integration standard
![Page 10: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/10.jpg)
An excess of unintegrated HIV DNA occurs off HAART
![Page 11: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/11.jpg)
An excess of unintegrated HIV DNA occurs in Elite Suppressors
![Page 12: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/12.jpg)
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
Co
pie
s p
er
cell
Total HIV DNAIntegrated HIV DNA
5.6
![Page 13: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/13.jpg)
One patient without an excess of unintegrated HIV DNA on HAART
![Page 14: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/14.jpg)
Sporadic excess of unintegrated HIV DNA occur on HAART
![Page 15: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/15.jpg)
Sporadic excesses of unintegrated HIV DNA on HAART
![Page 16: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/16.jpg)
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
![Page 17: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/17.jpg)
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
![Page 18: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/18.jpg)
Reproducibility total/integrated HIV
RU5/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
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Longitudinal samples suggest sporadic excess may occur
![Page 20: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/20.jpg)
Sporadic excess of unintegrated HIV DNA occur on HAART
![Page 21: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/21.jpg)
Sporadic blips of unintegrated HIV DNA on HAART
![Page 22: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/22.jpg)
Overtime total HIV DNA approaches integrated HIV DNA on HAART
Koelsch JID 2008
![Page 23: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/23.jpg)
The level of unintegrated HIV DNA decreases with HAART
19
1.5 1.4
6.5
2.8
3.01.0
2.3
Co
pie
s p
er
cell
![Page 24: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/24.jpg)
![Page 25: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/25.jpg)
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
cell
Total HIV (SST) Integrated HIV
11* 2.0 3.2 1.6 1.8 1.8 5.9 5.6
![Page 26: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/26.jpg)
The half-life of unintegrated HIV DNA is ~ 3 days in rCD4+T cells treated
![Page 27: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/27.jpg)
An excess of unintegrated HIV DNA occurs in some patients on HAART
![Page 28: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/28.jpg)
Assay of HIV integration
![Page 29: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/29.jpg)
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.
![Page 30: Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on HAART Una O’Doherty University of Pennsylvania.](https://reader035.fdocuments.us/reader035/viewer/2022062722/56649f2a5503460f94c43f67/html5/thumbnails/30.jpg)
Validation of our assay