Complete Transcriptome Analysis of Latently Infected CD4 + T Cells

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1 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

2 HIV-1 Latency in 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

3 In Vitro Model of HIV Latency in CD4 + T cells CD45R0 CD62L CCR7 CD25 2N 4N HLA-DR Ki67 gdna Marini et al. J Immunol. 181:7713, 2008

4 Microarray analysis of latently infected CD4 + T cells Sorted cells HIV-1 p24 Total culture Latently infected (p24+) Uninfected (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for microarray analysis RNA labeling Agilent 4 44k platform two-color competitive hybridization Iglesias-Ussel et al., Unpublished

5 Results of Microarray Analysis GO: Cell Activation, Proliferation, Survival GO: Gene Expression GO: Immune Function GO: Cell Metabolism Iglesias-Ussel et al., Unpublished

6 2ΔCt/average 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cells QRT-PCR Latently infected Uninfected % of Max Flow cytometry Infected Uninfected Iglesias-Ussel et al., Unpublished

7 Validation of higher CD2 expression in clinical samples Unsorted CD4 + T cells HLA-DR-APC SSC-A SSC-A Sorted Resting memory CD4 + T cells CD2 mid Sorted Resting memory CD4 + T cells CD2 high CD4-A700 CD45RA-APC-Cy7 CD2-PE Data generated by Claire Vandergeeten and Nicolas Chomont, VGTI-FL

8 Higher frequency of HIV DNA in resting CD2 high CD4 + T cells of HIV patients ST102 ST109 ST101 Tot HIV DNA (copies/10 6 cells) ST045 ST104 ST113 Data generated by Claire Vandergeeten and Nicolas Chomont, VGTI-FL

9 Higher recovery of viral particles from resting CD2 high CD4 + T cells of HIV patients Viral RNA (copies/10 6 cells) ST102* ST109* ST101 Viral RNA (copies/10 6 cells) Viral RNA (copies/10 6 cells) Insufficient cell recovery for ST045 * Low cell viability in the CD2 mid fraction Viral RNA (copies/10 6 cells) ST104 Viral RNA (copies/10 6 cells) ST113 Data generated by Claire Vandergeeten and Nicolas Chomont, VGTI-FL

10 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. CD2 bright is a marker of resting latently infected CD4 + T cells in vivo

11 Acknowledgments Institute of Human Virology Maria Iglesias-Ussel Alessandra Marini VGTI-FL Nicolas Chomont Claire Vandergeeten Funding NIAID-NIH Bill & Melinda Gates Foundation CHRB State of Virginia Johns Hopkins University Luigi Marchionni Hao Zhang

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