Transcriptomic Analysis of Peripheral Blood Mononuclear Cells in Rapid Progressors in Early HIV Infection Identifies a Signature Closely Correlated with Disease Progression Z.-N. Zhang, J.-J. Xu, Y.-J. Fu, J. Liu, Y.-J. Jiang, H.-L. Cui, B. Zhao, H. Sun, Y.-W. He, Q.-J. Li, and H. Shang August © Copyright 2013 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry Introduction  HIV infected rapid progressors (RPs) Represent 10-15% of the HIV-positive population Progress to AIDS within the first 1 to 3 years of HIV infection Associate with several viral, genetic and immunologic factors Remain less understood than those with chronic progression or elite controllers

© Copyright 2009 by the American Association for Clinical Chemistry Introduction  Host transcriptomic profiles associated with HIV disease progression mRNAs such as Interferon-stimulated genes have been reported to contribute to the distinct clinical outcomes of HIV infection Expression of several miRNA signatures, such as miR-29 family, miR-17/92 family were changed in HIV infected elite controllers or exposed seronegative individuals The pattern of transcriptomic profile associated with HIV disease progression remains largely elusive and the miRNA profiles of peripheral blood mononuclear cells (PBMCs) from RPs have yet to be examined

© Copyright 2009 by the American Association for Clinical Chemistry Question 1  Can a unique transcriptional signature that distinguishes HIV infected rapid progressors from chronic progressors be identified?

© Copyright 2009 by the American Association for Clinical Chemistry Methodology  Patient population -Rapid progressors (RPs): CD4<350 cells/ ul within 1 year of infection -Chronic progressors (CPs): CD4>500 cells/ul after 1 year of infection -PBMCs at 111±22 days (Mean±SD) of HIV infection  mRNA and miRNA microarray - mRNA: Roche NimbleGen platforms: transcripts - miRNA: SYBR-based real-time quantitative PCR: 347 miRNAs  miRNA overexpression and cell death detection - Nucleofection with pmaxGFP-miRNA vector - Annexin V and PI detection

© Copyright 2009 by the American Association for Clinical Chemistry Clinical characteristics of patients with early HIV infection and healthy controls RPs, rapid progressors CPs, chronic progressors HCs, healthy controls

© Copyright 2009 by the American Association for Clinical Chemistry Transcripts profile in RPs and CPs Figure 1. Transcripts differentially expressed in RPs compared with CPs and pathway enrichment analysis results. A) Principal component analysis (PCA, Partek software) was used to compare the gene expression signatures of individual persons in RPs with that of CPs. B) Hierarchical cluster analysis (Partek software) of the differentially expressed mRNAs in RPs compared with CPs with an FDR controlled P<0.05. C) The top ten pathways enriched in genes that were significantly upregulated in RPs compared with CPs with an absolute fold change of at least 1.5.

© Copyright 2009 by the American Association for Clinical Chemistry miRNA expression between RPs and CPs Figure 2. Differential miRNA expression between RPs and CPs. A) Five miRNAs differing significantly in the comparison of RPs and CPs in both training and validation cohort are shown. B) Hierarchical clustering of change in threshold cycle (DCt) of the five miRNAs differentially expressed between RPs and CPs in the validation cohort by complete linkage method and Pearson correlation.

© Copyright 2009 by the American Association for Clinical Chemistry Question 2  Can significant correlations be found in the altered expression of miRNAs and mRNAs in PBMCs of RPs ?

© Copyright 2009 by the American Association for Clinical Chemistry Figure 3. Combined analysis with mRNA and miRNA targets converged on enrichment to the apoptosis pathway. A) In the 391 significant up-regulated genes with a fold change of >50% within the RP transcriptome, 129 were the predicted targets of signature miRNAs. All upregulated predicted targets (from 5 miRNAs) with the 391 upregulated genes were put into pathway enrichment analysis. B) The upregulated mRNA expression and predicted signature miRNA targets converged on enrichment solely to the pathway of apoptosis and survival: Cytoplasmic/mitochondrial transport of proapoptotic proteins Bid, Bmf and Bim. C) Four genes were involved in this pathway and three of them were the predicted targets of the 5 miRNAs. Combined analysis with mRNA and miRNA enrichment to the apoptosis pathway

© Copyright 2009 by the American Association for Clinical Chemistry Figure 4. miR-31 overexpression promotes T cell survival. A) Representative data of CD4+T cell death ex vivo either with no stimulation or after TCR stimulation. The frequencies of cells are indicated as percentage of total CD4+GFP+ T cells. B) The proportion of Annexin V + cells or 7-AAD+ cells of CD4+T cells was significantly decreased by miR-31 overexpression compared with Mock. C) Representative data of CD8+T cell death ex vivo either with no stimulation or after TCR stimulation. The frequencies of cells are indicated as percentage of total CD8+GFP+ T cells. D) The proportion of Annexin V + cells or 7- AAD+ cells of CD8+T cells was significantly decreased by miR-31 overexpression compared with Mock. n=6. miR-31 overexpression promotes T cell survival

© Copyright 2009 by the American Association for Clinical Chemistry Question 3  Can the RP transcriptomal signature be used in HIV intervention?

© Copyright 2009 by the American Association for Clinical Chemistry Relationship between miRNA expression and disease progression Figure 5. Relationship between miRNA expression and disease progression Differential miRNA expression between RPs and CPs. A) The predictive capability of the 5-miRNA signature combination or individual miRNAs for rapid disease progression was calculated by ROC analysis. B) Kaplan–Meier survival analysis showed that the mean time for CD4+ T cell counts to reach <350 cells/μL in miRNA low expressers (miR-31, miR-200c, and miR-526a) was significantly shorter than that in high expressers.

© Copyright 2009 by the American Association for Clinical Chemistry  A 5-miRNA signature can serve as a biomarker to distinguish RPs from CPs in their early infection.  Comprehensive analysis of RP’s transcriptome revealed dysregulation in the apoptosis pathway.  Overexpression of one key miRNA, miR-31, promoted primary human T cell survival. Conclusions

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