evaluating residual hiv reservoirs
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Evaluating residual HIV reservoirs. Background or why is this important? What can be measured? Where should we measure it? Conclusions. Why study HIV persistence?. To understand the obstacles to HIV eradication (absolute or functional) - PowerPoint PPT PresentationTRANSCRIPT
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Evaluating residual HIV reservoirs
• Background or why is this important?• What can be measured?• Where should we measure it?• Conclusions
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Why study HIV persistence?
• To understand the obstacles to HIV eradication (absolute or functional)
• To test whether reducing residual viral levels further restores patient health– Life expectancy of a 20 year old with HIV getting
effective ART is 11 years less than a similar HIV neg person (The Antiretroviral Therapy Cohort Collaboration, Lancet, 2008)
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What markers of viral persistence can be measured?
Adapted from Furtado et al NEJM 1999
Promoter Proximal
transcripts
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What markers of viral persistence can be measured?
Adapted from Furtado et al NEJM 1999
Promoter Proximal
transcripts
Cell-free(plasma) HIV RNA
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What markers of viral persistence can be measured?
Adapted from Furtado et al NEJM 1999
Promoter Proximal
transcripts
Cell-free(plasma) HIV RNA
HIV DNAtotal,
Integrated,2LTR circles
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What markers of viral persistence can be measured?
Adapted from Furtado et al NEJM 1999
Promoter Proximal
transcripts
HIV DNA:total,
Integrated,2LTR circles
Cell-free(plasma) HIV RNA
Cell associatedHIV RNA:Promoter-proximal,
Multiply spliced,Unspliced.
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Total HIV DNA can be reliably detected and measured
Viard et al AIDS 2004
• Usually PCR-based assays
• can be performed on PBMC or purified cellular subsets
• doesn’t distinguish defectivefrom replication competentor recent from remote infection
• abundant evidence that valuesare biologically relevant
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Integrated can be measured and unintegrated DNA can be calculated from total HIV DNA
Koelsch et al JID 2008 Agosto Virology 2010
• assays for integrated HIV DNA are more cumbersome, less sensitive• don’t distinguish between replication competent/defective• unintegrated forms more likely to represent recent infection
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2LTR circles as a markerfor ongoing replication
Buzon et al, Nature Med, 2010
Subset of subjects showed a transient increase in 2LTR circles during Raltegravir intensification.
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TTM TEM
TNTTD
E
TCM
Th1,2,17, Treg
T-cell subsets support HIV persistence variably
CD45RA,CCR7
CD45RO, CCR7, CD27
CD45RO, CD27
CD45RO
CD45RA, CD45RO
• TCM and TTM account fora large majority of infected
cells.
• At lower CD4 counts, fractionof TTM increases and TCM Decreases (not shown)
Chomont et al Nat Med 2009
T0
***
N=17
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Cell associated infectivity
Treatment in acute and early infection results in more rapid clearance of infected cell reservoir and smaller residual reservoir size.
Chun et al JID 2007Finzi et al Nat Med 1999
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Cell associated infectivity
Treatment in acute and early infection results in more rapid clearance of infected cell reservoir and smaller residual reservoir size.
Stage of disease at initiation of ART affects Reservoir size. From Strain et al, JID 2005
Agrawal et al, MOLBPE013New “cellular infectious viral load
Assay”; 6th IAS HPTP
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Cell free and cell associated HIV RNA
– Residual viremia at very low levels (Schockmel JAIDS 1997, Dornadula JAMA 1999, Havlir JAMA 2001, Palmer JCM 2003, Palmer PNAS 2008, Hatano AIDS 2010, Chun JID 2011)
• Production +/- residual viral replication– Cell associated HIV RNA (Wong PNAS 1997, Lewin JVirol 1999, Gunthard
JID 2001, Fischer JID 2004)
• Decays over time but frequently remains detectable• Production +/- residual viral replication (Pasternak
MOPE075, 6th IAS HPTP)• Some transcription patterns more suggestive of
latent infection (Adams PNAS 1994, Li Jvirol 2003, Lassen PLoS Path 2006, Tyagi Jvirol
2010)
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Residual plasma viremia is detectable in most subjects on ART
Chun et al JID 2011 Palmer et al PNAS 2008
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Unspliced viral RNA is detectable in most patients starting ART after established HIV infection but is lower when ART is started during acute infection
Schmid et al PLoS One, 2010Furtado et al NEJM, 1999
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T cell stores in tissues
Blood25% extracellular fluid volume
2% of T lymphocytes
GALT60% of T lymphocytes
Everything else35-40% of T lymphocytes
Activating environmentHigh levels of CCR5 expression
High levels of HIV replication before ART
Poles, Anton, Markowitz, Lafeuillade, Chun,Rouzioux, Dandekar
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HIV infection frequency exceeds infection frequency of PBMC at multiple gut sites
• 8 HIV+ subjects on ART, VL <50 c/ml for a median 5 years
• Colonoscopy, upper EGD w/6 to 9 biopsies per site.
• HIV DNA measured from cell isolates and normalized by flow cytometry data for CD4+ Tcells.
• Extrapolated to total body cell numbers
≈1.2 x 109 infected CD4+ T cells≈ 1.2 x 107 latently infected cells
Yukl et al JID Nov 2010
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HIV infection frequency exceeds infection frequency of PBMC at multiple gut sites
• 8 HIV+ subjects on ART, VL <50 c/ml for a median 5 years
• Colonoscopy, upper EGD w/6 to 9 biopsies per site.
• HIV DNA measured from cell isolates and normalized by flow cytometry data for CD4+ Tcells.
• Extrapolated to total body cell numbers
≈1.2 x 109 infected CD4+ T cells≈ 1.2 x 107 latently infected cells
Yukl et al JID Nov 2010
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HIV RNA expression ishigher at all gut sites than in PBMC
• HIV RNA measured from cell isolates and normalized to GAPDH measures and flow
cytometry data
• Normalized to HIV DNA content, the per-infected celltranscriptional level is not
higher in gut Yukl et al JID Nov 2010
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Assessment of viral load on single cell level
Schacker et al JID, 2005
Can reveal information on nature of infected cells not obtained by bulk measurements
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Conclusions
What is the relevant form to measure?– For many questions, total HIV DNA, residual
plasma viremia are still powerful tools– Analyzing specific DNA and cell-associated
RNA forms can provide additional information about different forms of persistence and how they respond to interventions.
– Need to look at T-cell (and monocyte macrophage) specific viral load
– Need to study these cells in relevant tissues
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Conclusions
• In gut tissues, viral loads respectably high and are amenable to current generations of sensitive molecular assays
• Improvement in “high throughput technologies” may enable assessment of a larger sample of clinical materials
• Need for assessment on a single cell level: improved imaging techniques/ single cell analysis techniques
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Acknowledgements
SF VAMC/UCSF• Steven Yukl• Satish Pillai• Peilin Li• Harry Lampiris• Amandeep Shergill• Ken McQuaid
SFGH/UCSF• Diane Havlir• Steve Deeks• Hiroyu Hatano• Peter Hunt
UCSD• Matt Strain• Doug Richman• Susan Little• Karole Ignacio
U of Zurich• Huldrych Gunthard• Marek Fischer• Sara Gianella
Dept Exp Med/UCSF• Lorrie Epling• Elizabeth Sinclair
U Minn• Ashley Haase• Tim Schacker• Qing Sheng Li
Patient volunteers
Funding:Dept of Veterans AffairsNIHUCSF CFAR
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Persistently abnormal T-cell activation remains in ART treated patients and in elite controllers (viral load below LOD)
Hunt et al JID 2008
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HIV transcription patterns