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Transcription Profiling of CD4+ T Cells in Rhesus Macaques that Infected with Simian -Human Immunodeficiency Virus and Rechallenged with SIVmac251 5th World Congress on Virology Dec 07-09, 2015 Atlanta Hye Kyung Chung Ph.D Advanced BioScience Laboratories, Inc Viral Load SHIV infection Patterns Post-Infection 2 THANK YOU Advance BioScience Laboratories National Cancer Institute, NIH John Brady Ranajit Pal Lindsay Gregory Michael Lee Allison Younkins Ahae Woo Gayatri Patel Cindy Masision Mike Rodonovichi Moffitt Cancer Center , University of South Florida EunMi Lee Jessica Livesay Anthony Cristillo Gerald Kovacs Jae K. Lee Annamalai Muthiah Soo-Young Cheon Animal Facility Debora Weiss Jim Treece • Support: Partial Support by NIH/NIAID contract N01-AI-15430 to Advanced BioScience Laboratories Purpose • We hypothesize that there are differences among animals in their ability to control virus infection by mounting appropriate immune responses. • We believe that the molecular basis for these differences can be determined by analyzing gene expression profiles. • To test this hypothesis, we are comparing global gene expression patterns in rhesus macaques that control –or do not control- persistent virus replication. In vaccine research, we need to have better markers to predict whether a vaccine will be protective. Using the power of modern genetic analysis such as microarray, we can establish a “signature” or gene expression profile that defines the protective immune response. This technology has been used successfully to establish gene expression signatures, ALL-AML class Prediction in tumors. Science, 1999 5 Contents Part 1 Innate Host Immune Responses: Natural Control of SHIV Replication Part 2 Innate and Adaptive Antiviral Immune Responses Biomarkers of Innate and Adaptive Immunity in CD4+ T Cells 6 Part 1 Innate Immunity • An ideal vaccine stimulates four components of the immune system. 1) Elicit neutralizing antibody at high titer. 2) Stimulate a cellular (T-Cell) immune response, especially cytotoxic T-cells. 3) Stimulate mucosal immunity. 4) Provoke the innate immune system. • The rhesus macaque has served as an animal model system for vaccine development and also to study pathogenesis of several human infectious diseases. • Microarray technology has been extensively used to analyze a global gene expression pattern in cancer and other diseases to monitor the molecular mechanism of pathogenesis. • Knowledge of natural host defense mechanisms may lead to their exploitation for therapeutic or prophylactic purposes. 7 SHIVsf162P3 RNA levels in Plasma in Virus Infected Rhesus Macaques Viral RNA Copies/ml Plasma 108 713L 714L 717L 718L 719L 720L 68M 69M 107 106 108 107 106 105 105 104 104 103 103 0 20 40 60 80 100 Weeks Post Challenge 120 140 0 10 20 30 40 Days Post Challenge Chung, et al., Viral Immuno. 2008 50 Gene Expression Profiles in PBMC from Naïve vs SHIVsf162P3 infected Rhesus Macaques Condition Animal Time Controlled 714L, 718L Day 21 High 714L, 718L >Year 2 Low 713L, 720L Day 21 High 713L, 720L >Year 2 Non-controlled Naïve Viral load High Pool of 8 uninfected monkeys 9 Clustering Heatmap of Differentially Expressed Genes Day 21 Year 2 B. Year A. Day A2N713Y AN720Y A2C714Y 200648_s_at 204351_at 204860_s_at 203922_s_at 201670_s_at 205237_at 206715_at 210029_at 212335_at 209970_x_at 204924_at 207857_at 204588_s_at 202609_at 206343_s_at 215223_s_at 211367_s_at 212820_at 216841_s_at 212268_at 212224_at 209499_x_at 206710_s_at 211404_s_at 212192_at 203535_at 203041_s_at 203574_at 209230_s_at 212636_at 218728_s_at 201647_s_at 215001_s_at 202241_at 222156_x_at 202388_at 204533_at 217502_at 202411_at 214453_s_at 211368_s_at 205552_s_at 205660_at 206420_at 209969_s_at 202269_x_at 205483_s_at 201739_at 205842_s_at 201616_s_at 213516_at 214894_x_at 215594_at 221225_at 204838_s_at 212289_at 217340_at 207979_s_at 213109_at AC718Y AN720D A2N713D AC718D A2C714D 207979_s_at 204838_s_at 214894_x_at 201616_s_at 215594_at 217340_at 212289_at 221225_at 213109_at 213516_at 205660_at 211368_s_at 205483_s_at 205552_s_at 209969_s_at 202269_x_at 214453_s_at 202411_at 204533_at 217502_at 212636_at 202241_at 218728_s_at 209970_x_at 206715_at 211367_s_at 210029_at 201739_at 204924_at 222156_x_at 205842_s_at 202388_at 215001_s_at 203574_at 204351_at 206420_at 203041_s_at 201647_s_at 202609_at 206710_s_at 204588_s_at 212192_at 212224_at 212820_at 212335_at 212268_at 201670_s_at 200648_s_at 204860_s_at 216841_s_at 206343_s_at 209499_x_at 209230_s_at 205237_at 211404_s_at 203535_at 203922_s_at 207857_at 215223_s_at Chung, et al., Viral Immunol. 2008 Heatmap of Differentially Expressed Genes at Day 21 and Year 2 C. Day+Year A2N713Y AN720Y AN720D A2N713D A2C714Y AC718Y AC718D A2C714D 209970_x_at 204924_at 211367_s_at 201739_at 210029_at 206715_at 204533_at 217502_at 209969_s_at 202269_x_at 202411_at 214453_s_at 205660_at 211368_s_at 205552_s_at 205483_s_at 218728_s_at 215001_s_at 202388_at 202241_at 203574_at 212636_at 222156_x_at 206420_at 205842_s_at 206710_s_at 202609_at 204588_s_at 203041_s_at 201647_s_at 204860_s_at 216841_s_at 201670_s_at 200648_s_at 212224_at 212268_at 204351_at 212820_at 212335_at 206343_s_at 203922_s_at 207857_at 215223_s_at 209499_x_at 212192_at 209230_s_at 205237_at 211404_s_at 203535_at 204838_s_at 214894_x_at 207979_s_at 201616_s_at 215594_at 221225_at 213516_at 212289_at 217340_at 213109_at Chung, et al., Viral Immuno. 2008 Part 1 Summary We were examined differences between protective and non-protective host response by measuring global cellular gene expression profiles in SHIVinfected macaques. Comparing gene expression profiles in PBMC from animals that exhibit different levels of virus control showed interesting differences in gene expression pattern. We identified 59 genes. Activation of host defense-related genes such as interference-inducible genes, apoptosis genes, and inflammation-related genes are up-regulated in the non-controllers. The results suggested that these genes might contribute to a favorable microenvironment for virus replication in vivo. It could help identify early parameters that predict the efficiency of protective immune responses. 12 Part 2 Innate and Adaptive Immune Responses 0 5.2 y Group 1 N=3 SHIVsf162P3 0 SIV mac 251 Day 3 Day10 Day 28 Day 160 Day 3 Day 10 Day 28 Day 60 2.7 y Group 2 N=3 SHIVBaL SIV mac251 13 Characteristics of Rhesus Macaques Used for Global Gene Expression Analysis Animal* Weight 714L 717L 718L 184M 185M 186M 8.9 10.8 11.2 8.7 8.7 8.4 Sex Male Male Male Male Male Male Prior to SIV mac251 Challenge SHIVsf162P3 SHIVsf162P3 SHIVsf162P3 SHIV BaL SHIV BaL SHIV BaL Re-Challenge Virus SIVmac251 SIVmac251 SIVmac251 SIVmac251 SIVmac251 SIVmac251 Challenge Route Rectal Rectal Rectal Rectal Rectal Rectal 14 MHC Class 1 Genotyping Animal ID 714L 717L 718L 184M 185M 186M Mamu-A*01a Negative Negative Negative Negative Negative Positive Mamu-B*08b Negative Positive Negative Negative Negative Negative Mamu-B*17b Negative Negative Negative Negative Negative Negative 15 TRIM 5 alpha Genotyping G/T Animal T307 P327 Q332 Splice Site ID P T R Mutation 714L T/P P/T Q G/G 717L T/P P/T Q G/G 718L G/T T P Q 184M P P Q G/G P P Q 185M G/T 186M T/P P Q G/G Moderate Resistance 333 ACGTTT C1002A 334P A/S/ Genotypesa /Q/R T TF339-340 P341Q A/S P TRIM TFP/TRIM Q TF/∆∆ P/Q A/S P TRIM TFP/TRIM Q TF/∆∆ P/Q TRIM TFP/TRIM CYPA A P TF P A/S P TRIM TFP/TRIM Q TF/∆∆ P/Q S P TRIM Q/TRIM CYPA ∆∆ Q A P TRIM TFP/TRIM TFP TF P Most Resistance 16 Virological Outcome in Plasma/PBMC at Preand 160 days Post-Rechallenge Animal 714L 717L 718L 184M 185M 186M Prior to SIV mac251 Re-challenge Viral RNA SIV proviral DNA copy Load/ml of number / 106 PBMC Plasma <10 <50 <10 <50 3.37E+01 <50 1.53E+02 <50 5.39E+02 <50 3.29E+02 <50 160 days Post Challenge Viral RNA SIV proviral DNA copy Load/ml of number / 106 PBMC Plasma 1.03E+02 509 7.87E+01 <50 1.58E+02 64,192 3.52E+02 346 4.85E+03 61,638 2.45E+02 23,635 17 Viral RNA Levels in Plasma in SIVmac251 Infected Macaques 106 108 Viral RNA Load (Copies/ml) Viral RNA Load (Copies/ml) 184M 185M 186M 107 106 105 104 103 714L 717L 718L 105 104 103 102 102 101 101 0 20 40 60 80 100 Days Post Infection 120 140 160 180 0 20 40 60 80 100 120 140 160 180 200 Days Post Infection 18 Chung, et al., J Med Primatol. 2015 Heatmap for Differentially Expressed Genes between Protected and Un-Protected Rhesus Macaque 19 Heatmap for Differentially Expressed Genes with Human and Rhesus Macaque Chips 20 Network Diagram Derived from Eight Common Genes of Human Chip Microarray Data 21 Network Diagram Derived from Eight Common Genes of SIV mac251 Re-Challenge Study using Monkey Chip Microarray Data 22 Eight Protective Biomarker Human chip Monkey chip Exponential fold changes Exponential fold changes Gene Symbol Probe Set ID MX1 202086_at 2.61 2.23 IFI27 202411_at 6.83 2.23 S100A9 203535_at 5.33 1.18 TYROBP 204122_at 2.34 1.30 LMO2 204249_s_at 1.71 1.32 MX2 204994_at 2.10 1.77 FCN1 205237_at 4.35 1.52 JAK2 205842_s_at 2.35 1.74 23 Part 2 Summary Our finding suggested that SHIV a model of attenuated viral vaccineinduced protective immune response from pathogenic SIVmac 251 infection. Down regulation of certain interferon responses and tyrosine kinase pathway may indicate protective vaccine responses. The eight common genes MX1, MX2, IFI27, JAK2, LMO2, TYROBP, FCN1, and S100A9 with P values <0.01 were identified as a potential protective biomarker, and all genes were down-regulated in the protected macaques. 24 Conclusions Carefully designed global gene expression profiling could help identify early parameters that predict the protective immune responses in HIV vaccine evaluation in NHP model. Despite the fact that we were able to identify statistically significant differences in expression levels between protected and unprotected animals these observations were warrant validation using larger study groups. 25 THANK YOU Advance BioScience Laboratories National Cancer Institute, NIH John Brady Ranajit Pal Lindsay Gregory Michael Lee Allison Younkins Ahae Woo Gayatri Patel Cindy Masision Mike Rodonovichi Moffitt Cancer Center , University of South Florida EunMi Lee Jessica Livesay Anthony Cristillo Gerald Kovacs Jae K. Lee Annamalai Muthiah Soo-Young Cheon Animal Facility Debora Weiss Jim Treece • Support: Partial Support by NIH/NIAID contract N01-AI-15430 to Advanced BioScience Laboratories
