“Towards an HIV Cure”
Pre-Conference Symposium
20 & 21 July 2012
Complete Transcriptome Analysis
+
of Latently Infected CD4 T Cells
Fabio Romerio
Institute of Human Virology
University of Maryland School of Medicine
Baltimore, MD - USA
HIV-1 Latency in
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+
CD4
T cells
Resting CD4+ T cells are the largest reservoir of latent provirus
Established during primary infection, replenished continuously
Produce some viral RNA, but no viral proteins and no virions
Latent HIV is refractory to HAART, invisible to immune system
Major obstacle to cure
Extremely rare in peripheral blood
Indistinguishable from uninfected cells
Development of in vitro models:
 Technical expediency vs. physiological relevance
 Achievement of true viral latency and cellular quiescence
 Validation with clinical samples
CD45R0
In Vitro Model+of HIV Latency
in CD4 T cells
CD62L
CCR7
CD25
2N 4N
HLA-DR
Marini et al. J Immunol. 181:7713, 2008
Ki67
gDNA
Microarray analysis
of
latently
infected CD4+ T cells
Sorted cells
Total culture
Latently infected
(p24+)
Uninfected
(p24–)
HIV-1 p24
• Quiescent HIV-1 positive and HIV-1 negative
cells were sorted from the same culture (4
different donors) after intracellular staining
with anti-p24 antibodies
FSC-H
RNA extraction
• Total RNA was extracted from the two cell
populations, labeled with Cy3 and Cy5 (dye
swap) and used for microarray analysis
RNA labeling
Agilent 4×44k platform
two-color competitive
hybridization
Iglesias-Ussel et al., Unpublished
Results of Microarray Analysis
GO: Cell Activation, Proliferation, Survival
GO: Gene Expression
GO: Immune Function
GO: Cell Metabolism
Iglesias-Ussel et al., Unpublished
Higher expression of CD2 on
latently infected cells
• We found 33 transcripts for surface markers differentially expressed on
latently infected vs. uninfected cells (20 up-regulated, 13 down-regulated)
• For one of these receptors (CD2) we have validated differential expression
in in vitro-generated latently infected vs. uninfected cells
QRT-PCR
Flow cytometry
Latently infected
Infected
Uninfected
1.0
0.5
% of Max
2ΔCt/average
1.5
0.0
Iglesias-Ussel et al., Unpublished
Uninfected
Validation of higher CD2
expression in clinical samples
SSC-A
SSC-A
HLA-DR-APC
Unsorted
CD4+ T cells
Sorted
Resting memory
CD4+ T cells CD2mid
Sorted
Resting memory
CD4+ T cells CD2high
CD4-A700
CD45RA-APC-Cy7
CD2-PE
Data generated by Claire Vandergeeten and Nicolas Chomont, VGTI-FL
Higherhigh
frequency
of HIV DNA in resting
+
CD2 CD4 T cells of HIV patients
Tot HIV DNA (copies/106 cells)
ST102
ST045
ST109
ST104
Data generated by Claire Vandergeeten and Nicolas Chomont, VGTI-FL
ST101
ST113
Insufficient cell
recovery for ST045
* Low cell viability
in the CD2mid
fraction
ST104
Viral RNA (copies/106 cells)
ST109*
Viral RNA (copies/106 cells)
Viral RNA (copies/106 cells)
ST102*
Viral RNA (copies/106 cells)
Viral RNA (copies/106 cells)
Higher recovery
of
viral particles from resting
high
+
CD2 CD4 T cells of HIV patients
Data generated by Claire Vandergeeten and Nicolas Chomont, VGTI-FL
ST101
ST113
Conclusions
• In vitro latency model achieves cell quiescence and viral latency
• Microarray analyses reveal profound differences of gene expression
between latently infected and uninfected cells
• Cellular environment conducive to cell quiescence and viral latency:
 phenotype induced by HIV vs. phenotype selected by HIV?
• Down-modulation of several cell functions (gene expression, cell
activation and proliferation, cell metabolism) halt HIV replication:
 implications for viral eradication with current anti-latency drugs
• Differential expression of transcripts for 33 surface markers
• Increased surface expression of CD2 was validated on latently
infected cells generated in vitro and also in clinical samples
 High levels of CD2 define a subset of resting CD4+ T cells carrying
higher frequency of replication competent HIV provirus.
 CD2bright is a marker of resting latently infected CD4+ T cells in vivo
Acknowledgments
Institute of Human Virology
• Maria Iglesias-Ussel
• Alessandra Marini
VGTI-FL
• Nicolas Chomont
• Claire Vandergeeten
Johns Hopkins University
• Luigi Marchionni
• Hao Zhang
Funding
•NIAID-NIH
•Bill & Melinda Gates
Foundation
•CHRB – State of Virginia