sporadic excesses of unintegrated hiv dna can occur in hiv+ patients on haart
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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Sporadic excesses of unintegrated HIV DNA can occur in HIV+ patients on
HAART
Una O’DohertyUniversity of Pennsylvania
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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
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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.
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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
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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
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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)
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Validation of our assay
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Validation of integration assay
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Confidence interval: ratio of total HIV DNA/integrated with our integration standard
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An excess of unintegrated HIV DNA occurs off HAART
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An excess of unintegrated HIV DNA occurs in Elite Suppressors
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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
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One patient without an excess of unintegrated HIV DNA on HAART
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Sporadic excess of unintegrated HIV DNA occur on HAART
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Sporadic excesses of unintegrated HIV DNA on HAART
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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
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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
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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
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Longitudinal samples suggest sporadic excess may occur
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Sporadic excess of unintegrated HIV DNA occur on HAART
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Sporadic blips of unintegrated HIV DNA on HAART
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Overtime total HIV DNA approaches integrated HIV DNA on HAART
Koelsch JID 2008
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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
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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
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The half-life of unintegrated HIV DNA is ~ 3 days in rCD4+T cells treated
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An excess of unintegrated HIV DNA occurs in some patients on HAART
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Assay of HIV integration
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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](https://reader036.vdocuments.mx/reader036/viewer/2022062814/5681678d550346895ddcb1d9/html5/thumbnails/30.jpg)
Validation of our assay