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  • 1. Thinking about HIV: the intersection of virus, neuroinflammation and cognitive dysfunction2. Immune privilege and HIV-1 persistence in the CNS
  • So to address our question, we need to isolate viral sequences from our patients and compare them to clinical parameters indicative of disease progression. To do this we draw whole blood from patients participating in our cohort, which then enters our BSL-3 facility where blood cells are separated from the serum. These cells are lysed and we PCR amplify the proviral DNA, which is then subject to gel electrophoresis we send the product for sequencing analysis to identify SNPs, as well as functional analysis to quantify transcription activation.
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  • So far we have a total of 91 brain samples: 15 normal, 46 minor cognitive motor disorder, 30 HADEarly trends show there is in fact a correlation between genetic variation with the LTR and neurocognitive statusa higher frequency of base pair changes in spleen derived tissue as compared to the brain 3T/4G compartmentalizes in specific brain regions such as Thalamus but and increases prevalence with disease severityOpp trend observed in 3T/6G which compartmentalizes with cerebellum but decreases in prevalence with disease severity

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  • 1. Viral Promoter Polymorphisms in HIV Disease Gregory C. Antell 2013 Sigma Xi Research Showcase March 15, 2013 Graduate Student Drexel UniversitySchool of Biomedical Engineering, Science, and Health Systems
  • 2. The HIV epidemic has neurological consequences An average of 6,800 new HIV infections and 5,700 HIV-related deaths occur daily worldwide Infection of the central nervous system occurs in approximately 80% of infected individuals Approximately 50% of HIV-infected adults and children will demonstrate a neurological disorder at one time The advent of anti-retroviral therapy has diminished the incidence of HIV-associated neurocognitive disorders to a lesser extent than other AIDS-related diseases Prevalence of neurocognitive diseases has actually increased due to the prolonged survival of HIV infected individuals Neuropathology of HIV disease remains largely unknown and a critical area of current and future researchINSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 3. Multiple factors influence HIV-1 pathogenesis and HIV-1- associated neurocognitive disorders (HAND) Host & Therapy Cellular Pathogenesis is shaped A spectrum of cellular targets are by the host immune vulnerable to infection, which may lead to response and genetics, physiological compartmentalization and drug therapy, drug tissue-specific selective pressures. abuse, and aging. HANDViralMolecular diversity emerges in the virus as it adapts to selective HIV-associated neurocognitivepressures. Particular variants may disorders include HIV-associated serve as biomarkers of advanced dementia (HAD) and minor neurological disease. cognitive motor disorder (MCMD). INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 4. HIV-1 pathogenesis and associated neurological dysfunction CNS viral evolution BrainBloodBrainBarrier CNS viral entry Extra-CNS viral evolution Blood HIV-1 likely enters the brain during acute infection and during the absence of effective therapy or immune dysfunction In the brain resident microglial cells and perivascular macrophages are the major cellular targets for infection Release of viral and cellular neurotoxic mediators results in the alteration of the blood-brain barrier and neuronal dysfunction Acute Infection Clinical Latency AIDS / Dementia CD4 count > 500 200-500 < 200 INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 5. HIV-1 CNS entry and infection of resident cell populations Peripheral Blood Brain Mucosal compartment HIV-1-infected perivascular macrophage Lymphoid compartment resident microglia cellsBone marrow compartment Viral gene products have neurotoxic effects on astrocytes and neurons Blood- Other end organs Brain Barrier astrocytes neurons INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 6. HIV-1 replication scheme HIV-1 replication is controlled by the viral promoter, termed the long terminal repeat (LTR), as well as the regulatory genes Tat and Vpr While HIV-1 is known to have an entry phenotype, it is hypothesized that it may also have distinct replication phenotypes that associates with particular host cell phenotypes and/or physiological compartments Absorption and entry gp120 CD4 Budding Coreceptors CCR5 CXCR4 genomic Assembly Reverse RNA Transcription Protein synthesis Nuclear Transport Viral Gene and processing and Integration Expression integrated proviral DNA mRNA INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 7. The HIV-1 genome and 5’-LTR organization tat vpr rev 5’LTR gag vif 3’LTR pol vpu env nef 455 552 638nt1 leader U3 R U5 nuc-0 HS2 HS3 +1 nuc-1 HS4 -405 -245 +20 +165 Modulatory region Core region Enhancer region The HIV-1 genome is flanked by two LTR sequences: the 5’-LTR and the 3’-LTR The 5’-LTR acts as the promoter for viral gene expression The LTR contains a high concentration of binding sites for cellular transcription factors, which can vary according to the host cell phenotype INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 8. Background and demographics of the DREXELMED HIV/AIDS Genetic Analysis CohortPatients enrolled in the DrexelMed Cohort are recruited from the PatientsPhiladelphia region and are scheduled to return every six months. Visit SeenAt each visit, a patient interview is conducted, a blood sample iscollected, and a neurological exam is performed. Initial Visit 503 First Return 298 Demographic Count (%)/Mean (+/- SD) Second Return 202 Categories Variables with clinical variables Male 332 (66%) Third Return 136Gender Female 169 (33.6%) Fourth Return 95 Black/AA 418 (83.1%) Fifth Return 67 White 62 (12.3%)Race Other (AI/AN, multiple, Sixth Return 43 16 (3.2%) asian) Seventh Return 29 Unknown 7 (1.4%) cH 424 (84.3%) Eighth Return 17HAART status dH 43 (8.5%) Ninth Return 7 nH 34 (6.7%) Tenth Return 2Age 45.43 (± 8.569)Years since diagnosed 11.916 (± 7.312) Total 1399 INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 9. Research Focus #1: Do specific HIV-1 LTR single nucleotide polymorphisms (SNPs) derived patient peripheral blood samples correlate with alterations in clinical HIV disease parameters in the HAART era?INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 10. Viromic analysis of DREXELMED HIV/AIDS Genetic Analysis Cohort in the HAART era Ficoll-Pacque Qiagen DNEasyWhole Blood Plus gradient Tissue Kit Serum & PBMC separation Luminex Human PCR amplify BSL-3 Facility Cytokine 30 plex proviral DNA Separate on Serum and cell banking agarose gel Clinical and virus/host genomic data management HIV-1 Sequence Database Sequence PCR product analysis sequencing Gel extraction pGL3 Basic pCR4-TOPO Incubate with PCR amplify/ Taq to add AFunctional overhang analysis clone proviral DNA INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 11. HIV-1 LTR SNP densities in patients from the DREXELMED HIV/AIDS Genetic Analysis Cohort 900 700 Sequence Coverage 800 SNP Density (Number of mutations) SNP Density 600Coverage (Number of Sequences) 700 500 600 500 400 400 300 300 200 200 100 100 0 0 115 381 400 134 153 172 191 210 229 248 267 286 305 324 343 362 419 438 457 476 495 514 533 552 571 590 609 628 20 39 58 77 96 1 Nucleotide Position on ConB (Jan 2002) Reference Sequence • LTR SNP coverage and frequency was calculated for 461 patients and 1127 sequences • SNPs are observed throughout the LTR sequence and can be mapped to transcription factor binding sites INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 12. Nine HIV-1 LTR SNPs associate with change in CD4 count and log viral load away from the average of the cohort  The single nucleotide polymorphisms (SNPs) identified from patient peripheral blood samples can be plotted according to base pair position in the LTR and association with CD4+ T cell count and log viral load  Data is adjusted for patient age, sex, and raceINSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 13. Significant LTR SNPsPhenotype Position Ref./Mut. Mutant Freq Effect p-value 108 A/CGT 38.0% -41.228 0.0176 120 C/AT 6.2% 72.950 0.0200CD4 Count 181 A/CG 8.3% -72.320 0.0173 293 G/ACT 11.4% -46.920 0.0452 108 A/CGT 38.0% 184.4% 0.0010 115 A/GT 18.5% 60.7% 0.0301Viral Load 160 C/AG 6.3% 46.7% 0.0278 168 G/ACT 14.8% 60.2% 0.0282 251 G/ACT 8.8% 53.9% 0.0315 A total of 9 SNPs, located at 8 distinct nucleotide positions, were identified to associate with the clinical parameters of CD4+ T cell count and/or viral load at a statistically significant level (p-vale < 0.05). The effect in this case is defined as the change in CD4+ T cell count or the percent change in viral load away from the average.INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 14. Research Focus #2: Are these significant peripheral blood HIV-1 LTR single nucleotide polymorphisms (SNPs) also found in HIV-infected brains?INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 15. Isolation of HIV-1 brain-derived LTRs for sequence analysis Nested PCR amplifies LTRAutopsy tissue punches QIAGEN DNeasy from proviral DNA Tissue Procedure PCR Isolation of amplify genomic DNA proviral DNA HIV-1 Brain LTR Sequence Database Separate on agarose gel Preparation for sequencing and sequence analysis INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 16. Number of brain and spleen sequences used in analysisPatient samples available BrainHAD 6 Normal Subsyndromic MCMD HADMCMD 16 SequenceSubsyndromic 3 Number 16 18 95 38Normal 2 PatientUninfected 1 Number 2 2 14 6TOTAL 28 SpleenTissue regions available Normal Subsyndromic MCMD HADCerebellum 28 SequenceDeep White Matter 28 Number 3 2 19 7Head of Caudate 28 PatientMidfrontal Gyrus 28 Number 2 2 14 4Parietal 28Thalamus 27 Autopsy tissue samples were collected from multipleSpleen 23 brain sites, as well as spleen, from patients with varying degrees of neurological impairment. National NeuroAIDS Tissue Consortium – University of Texas Director: Ben Gelman, M.D., Ph.D. INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 17. Prevalence of clinically significant peripheral blood LTR SNPs in HIV-1 infected brain tissueNucleotide TF Number of Total in Total in Neuro. Neuro. Position Site individuals Spleen Brain Normal Impaired COUP/ 108 20 11 75 13 73 AP1 COUP/ 115 5 1 6 0 7 AP1 120 COUP 5 1 10 1 10 160 AP1 2 0 3 0 3 168 unk 8 1 10 0 11 181 unk 4 4 0 0 4 251 unk 10 4 13 0 17 293 USF 8 5 8 0 13 INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 18. Clinically significant peripheral blood HIV-1 LTR SNPs are found in all regions of the HIV-1-infected brain except for SNP 181 16 14 12Number of SNPs Cerebellum 10 Deep White Matter 8 Head of Caudate Midfrontal Gyrus 6 Parietal 4 Thalamus 2 0 108 115 120 160 168 181 251 293 LTR SNP Position INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 19. HIV-1 LTR SNPs identified in the peripheral blood are also found to associate with neurologic impairment in the brainNucleotide Found in TF Site Texas Cohort Notes DREXELMED PBMC Notes Position Brain? Decreases with Decreased CD4 count 108 Yes COUP/AP1 impairment Increased viral load 115 Yes COUP/AP1 Only in impairment Increased viral load 120 Yes COUP Mostly in impairment Increased CD4 count Rare, only found in 160 Yes AP1 Increased viral load brain and impairment 168 Yes unk Only in impairment Increased viral load 181 No unk Only found in spleen Decreased CD4 count 251 Yes unk Only in impairment Increased viral load 293 Yes USF Only in impairment Decreased CD4 count INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 20. Frequency of LTR position 108 polymorphism (A to G) with respect to neurocognitive status 100% 92% 100% 90% 90% 80% 70% 63% 60% 54% 50% 50% BRAIN 50% 46% 40% SPLEEN 30% 20% 10% 0% NORMAL SUBSYNDROMIC MCMD HAD BRAIN SPLEENNucleotide Normal Subsyndromic MCMD HAD Nucleotide Normal Subsyndromic MCMD HADA (reference) 1 1 24 13 A (reference) 0 1 7 2G (mutation) 11 9 40 15 G (mutation) 2 1 6 2 INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 21. Frequency of LTR position 168 polymorphism (G to A) with respect to neurocognitive status 25% 20% 18% 15% 10% 8% 8% 5% 0% 0% 0% 0% 0% BRAIN 0% SPLEEN BRAIN SPLEEN Normal Subsyndromic MCMD HAD Normal Subsyndromic MCMD HADG (reference) 12 10 54 24 G (reference) 2 2 10 4A (mutation) 0 0 10 4 A (mutation) 0 0 3 0 INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 22. Frequency of LTR position 251 polymorphism (G to A/C) with respect to neurocognitive status 25% 23% 20% 16% 15% 14% BRAIN 10% SPLEEN 5% 0% 0% 0% 0% 0% 0% NORMAL SUBSYNDROMIC MCMD HAD BRAIN SPLEEN Normal Subsyndromic MCMD HAD Normal Subsyndromic MCMD HADG (reference) 12 10 54 24 G (reference) 2 2 10 4A/C (mutation) 0 0 10 4 A/C (mutation) 0 0 3 0 INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 23. Brain-derived HIV-1 LTR vSNPs at positions 115, 120, 160, and 293 associated with neurocognitive impairment50% 50% SNP 115 SNP 12040% 40% A to G/T C to T30% 30% 25%20% 20% 11% 11% 8% 8% 7%10% 5% 10% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% NORMAL SUBSYNDROMIC MCMD HAD NORMAL SUBSYNDROMIC MCMD HAD50% 50% SNP 160 SNP 293 38%40% 40% C to A/G G to A/C30% 30%20% 20% 13%10% 5% 10% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%0% 0% NORMAL SUBSYNDROMIC MCMD HAD NORMAL SUBSYNDROMIC MCMD HAD INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 24. Summary of major findings• Eight HIV-1 LTR SNPs derived from peripheral blood mononuclear cells associate with change in CD4 count and/or log viral load away from the average of the cohort• Clinically significant peripheral blood HIV-1 LTR SNPs are found in all regions of the HIV-1-infected brain except for SNP 181• HIV-1 LTR SNPs identified in the peripheral blood are also found to associate with neurologic impairment in the brain, particularly SNPs 108, 168, and 251INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 25. Future directions• Identify SNPs in PBMC-derived LTR sequence that correlate with neurological disease and determine if they are present in HIV-1-infected brain tissue• Identify SNPs in brain-derived LTR sequences that associate with neurological impairment, and assess their presence in PBMC-derived LTRs• Analyze additional HIV genes that contribute to proviral transcription, such as Tat and Vpr, for single nucleotide polymorphisms that correlate with clinical parametersINSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 26. Ultimate objective of this research To identify a panel of genetic variants in the proviral HIV-1 LTR (or other parts of the genome) derived from PBMCs that are predictive of neurologic decline We envision a scenario where a simple blood test and diagnostic PCR can cue physicians about potential problems and treatment strategies. This viral SNP marker panel would be used in tandem with other neurocognitive biomarkers.INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE
  • 27. Brian Wigdahl, Ph.D., Professor & Chair Department of Microbiology & Immunology Drexel University College of Medicine DirectorWilliam Dampier, Ph.D. Olimpia Meucci, M.D., Ph.D. Betty Condran Renzo PeralesRui Feng, Ph.D. Sonia Navas-Martin, Ph.D. Jessica Cross Matt RimbeyJeffrey Jacobson, M.D. Michael Nonnemacher, Ph.D. Satinder Dahiya Germaine RivalPooja Jain, Ph.D. Vanessa Pirrone, Ph.D. David Downie Fiorella RossiSteve Jennings, Ph.D. Laura Steel, Ph.D. Brian Frantz Sonia ShahZafar Khan, Ph.D. Nirzari Parikh, M.S. Archana Gupta Luz Jeanette SierraSandhya Kortagere, Ph.D. Shendra Passic, M.S. Nneka Ikpeze Marianne StrazzaFred Krebs, Ph.D. Benjamas Aiamkitsumrit Shawn Keogan Gokul SwaminathanMichele Kutzler, Ph.D. Greg Antell Christina Kollias Ken ThompsonDavid Libon, M.D. Brandon Blakey Sharon Lewis Cristian ValenciaJulio Martin-Garcia, Ph.D. Jessica Brown Raphael Lukov Jean WilliamsBrian Moldover, Ph.D. Natalie Chen Andrea Partridge Wen Zhong NINDS NIMH NCI NIDA NIAID INSTITUTE FOR MOLECULAR MEDICINE AND INFECTIOUS DISEASE