sporadic excesses of unintegrated hiv dna can occur in hiv+ patients on haart una o’doherty...
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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
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
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
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)
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
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
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
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
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
19
1.5 1.4
6.5
2.8
3.01.0
2.3
Co
pie
s p
er
cell
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
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