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Project 3 SIV models of neurobehavioral, antiviral, and immunomodulatory SP antagonist(s) effects

Project 3 SIV models of neurobehavioral, antiviral, and immunomodulatory SP antagonist(s) effects. Andrew A. Lackner, DVM, PhD Kate Baker, PhD Tulane National Primate Research Center Tulane University Health Sciences Center. Project goals.

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Project 3 SIV models of neurobehavioral, antiviral, and immunomodulatory SP antagonist(s) effects

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  1. Project 3SIV models of neurobehavioral, antiviral, and immunomodulatory SP antagonist(s) effects Andrew A. Lackner, DVM, PhDKate Baker, PhD Tulane National Primate Research Center Tulane University Health Sciences Center

  2. Project goals • To determine the association between SIV progression, SP level, and indices of psychological disturbance. • Identify mechanisms whereby SP or SP antagonists affect SIV disease progression.

  3. Hypothesis • SP production will be increased in SIV-infected macaques and SP antagonists will have a protective effect on disease progression and neurobehavioral parameters by: • Decreasing SIV replication in monocyte/macrophages, particularly in the CNS. • Blocking the known neurobehavioral effects of SP.

  4. Specific Aims • Determine the cellular distribution of SP and its receptor in tissues of normal and SIV-infected macaques at various stages of disease. • Characterize the natural history of SP in normal and SIV-infected rhesus macaques in relation to viral, immunologic and behavioral measures. • Examine the effects of SP antagonists in normal and SIV-infected macaques. • Compare disease progression between continuous SP antagonist dosing with intermittent treatment (or periodically increased dose), timed to social rearrangements.

  5. Aim 1: Determine the cellular distribution of SP and its receptor in tissues of normal and SIV-infected macaques at various stages of disease (acute infection, clinically asymptomatic, terminal) Archival tissues from 24 infected and 24 uninfected macaques 8 acute infection + 8 matched controls 8 clinically asymptomatic/viral set point + 8 matched controls 8 terminal AIDS + 8 matched controls Detection of SP+cells by IHC and ISH Detection of NK1R+ cells by IHC and ISH Multiparameter confocal microscopy for cell type and virus (for SIV-infected animals) SP NK1R Macrophage SP NK1R NK cell SP NK1R T cell SP NK1R SIV Examination of SP and its receptors will focus on examining macrophages and T cells which may support viral replication and/or be involved in the adaptive or innate immune response (including NK cells). Multiple tissues will be examined but the focus will be on the CNS. Examination of cell type will be performed in the context of histopathologic lesions.

  6. Aim 2 • Characterize the natural history of SP in normal and SIV-infected rhesus macaques in relation to viral, immunologic and behavioral measures. • Prospectively examine i) levels of SP in blood and CSF over time, ii) viral load, iii) humoral and cellular immune responses and iv) natural killer cell activity. • Prospectively examine circadian rhythms and behavioral changes focusing on indices of psychological disturbance. This aim will establish the relationship between SP, viral load, behavior and immunologic parameters.

  7. Aim 3 • Examine the effects of SP antagonists in normal and SIV-infected macaques • Evaluation of the pharmacokinetics of SP antagonists. • Evaluate the effects of SP antagonists (for 6 months) in SIV-infected macaques for effects on viral load, viral dynamics and immunologic response compared to controls. • Evaluate the effects of SP antagonists on behavior. • Evaluate the effects of SP antagonists plus antiretrovirals (eg tenofovir) in SIV-infected macaques. This aim will be used to determine the pharmacokinetics, safety and potential efficacy of SP antagonists in macaques and serve as a guide for studies in HIV-infected humans described in project 4.

  8. SP, immunological, and behavioral measures * 16 SIV-pos. subjects Treated with SP antagonist 8 SIV-pos. subjects Untreated Aim 2:Characterize the natural history of SP in normal and SIV-infected rhesus macaques in relationship to viral, immunological, circadian, and behavior measures All subjects (N=32), single housing All subjects (N=32) introduced into pair housing SP, immunological, and behavioral measures * 24 subjects inoculated with SIV Longitudinal SP, viral, immunological, and behavioral measures * These data will serve as the baseline data for subsequent within-animal paired comparisons following infection and/or treatment with SP antagonists, as well as contributing data on normal untreated animals for Aim 2

  9. Aim 3a: Evaluation of the pharmacokinetics of SP antagonists 8 SIV-infected macaques (released from other studies) 8 normal uninfected macaques* Treat with SP antagonists Treat with SP antagonists Collect blood to measure -Antagonist -Viral load (for infected animals) -Serum chemistries • * will utilize 8 of the animals shown used in aims 2/3 either prior to infection or from the uninfected control group

  10. Aim 3:Examine the effect of SP antagonists in normal and SIV-infected macaques SP, immunological, and behavioral measures * Aim 3b and 3c Aim 2 8 SIV-neg. subjects Treated with SP antagonist 16 SIV-pos. subjects Treated with SP antagonist 8 SIV-pos. subjects Untreated To aim 3d To aim 4 Aim 2:Characterize the natural history of SP in normal and SIV-infected rhesus macaques in relationship to viral, immunological, circadian, and behavior measures All subjects (N=32), single housing All subjects (N=32) introduced into pair housing SP, immunological, and behavioral measures * 24 subjects inoculated with SIV Longitudinal SP, viral, immunological, and behavioral measures for 6 months * These data will serve as the baseline data for subsequent within-animal paired comparisons following infection and/or treatment with SP antagonists, as well as contributing data on normal untreated animals for Aim 2

  11. Aim 3d Evaluate the effects of SP antagonists plus antiretrovirals (eg tenofovir) in SIV-infected macaques on viral load, immunologic response and behavior compared to SIV-infected macaques treated with SP antagonists alone. 16 SIV-pos. subjects Treated with SP antagonist 8 SIV-pos. subjects Treated with SP antagonist plus tenofovir 8 SIV-pos. subjects Treated with SP antagonist Longitudinal SP, viral, immunological, and behavioral measures

  12. Schedule of animal monitoring *General health check, food consumption and stool character are recorded daily

  13. Kinetics of SIV-specific CTL responses in early SIV infection Gated through CD3+CD8HI lymphocytes Intestinal lymphocytes Peripheral blood lymphocytes mm352 mm353 mm356 mm352 mm353 mm356 0.2% 0.1% 0.1% 0.0% 0.0% 0.0% 7 days p.i. 20% 3.1% 8.7% 14.7% 4.1% 10.5% 17.3% 14 days p.i. 1.3% 1.8% 5.3% 1.8% 3.4% 11.4% 21 days p.i. 22.3% 8.5% 11.3% 4.6% 4.5% 3.0% 63 days p.i. 20% Mamu-A*01/p11C, C-M

  14. SIV-gag tetramer in the CNS parenchyma of an animal with SIVE

  15. Human (P < .001) 90 60 Substance P (pg/ml) Rhesus Macaque (P = .041) 30 0 HIV- (n=51) HIV+ (n=65) SIV- (n=4) SIV+ (n=8) Elevations in SP in humans infected with HIV and macaques infected with SIV

  16. Decreased NK cell activity during SIV infection LU20/107PBMC

  17. Specific Aims • Determine the cellular distribution of SP and its receptor in tissues of normal and SIV-infected macaques at various stages of disease. • Characterize the natural history of SP in normal and SIV-infected rhesus macaques in relation to viral, immunologic and behavioral measures. • Examine the effects of SP antagonists in normal and SIV-infected macaques. • Compare disease progression between continuous SP antagonist dosing with intermittent treatment (or periodically increased dose), timed to social rearrangements.

  18. The macaque model is ideally suited to this project • The rhesus macaque is the premier animal model for the study of AIDS neuropathogenesis. • The rhesus macaque has a long history of being employed to model psychopathology -- a well-characterized model of indices of psychological disturbance.

  19. Neurobehavioral measures • Telemetry data: circadian rhythms, sleep, activity level • Behavioral observation: • Species-appropriate social and non-social behavior • Abnormal behaviors • Psychological indices of disturbance

  20. Primate models of psychological disturbance: Depressive behaviors • Operational Definition • Slumped or collapsed body posture • Periods of unresponsivity to environmental stimuli • Periods of hypervigilance • Ethopharmacologic validation • Amelioration associated with tricyclics and MAO-I inhibits • Exacerbated with reserpine • Construct validation (causes and physiological correlates) • Associated with social stress as in humans • Associated with altered HPA and dopaminergic function as in humans • Face validity: Observational characteristics in humans • Slumped or collapsed body posture • Unresponsivity to environmental stimuli

  21. Primate models of psychological disturbance: Anxiety-related behaviors • Operational Definition • Self-directed ‘displacement’ behaviors • Scratching • Self-grooming • Body shaking • Ethopharmacologic validation • Amelioration (dose dependent) with lorazepam, midazolam, diazepam, clonidine • Exacerbated with axiogenics (FG142, b-CCE) • Construct validation (causes and physiological correlates) • Associated with uncertainty and anticipation of aversive conditions as in humans • Associated with autonomic hyperarousal as in humans • Face validity: Observational characteristics in humans • Self-directed ‘displacement’ behaviors • Scratching • Self-grooming

  22. Aim 3:Examine the effect of SP antagonists in normal and SIV-infected macaques SP, immunological, and behavioral measures Aim 3b and 3c Aim 2 8 SIV-neg. subjects Treated with SP antagonist 16 SIV-pos. subjects Treated with SP antagonist 8 SIV-pos. subjects Untreated Aim 2:Characterize the natural history of SP in normal and SIV-infected rhesus macaques in relationship to viral, immunological, circadian, and behavior measures All subjects (N=32), single housing All subjects (N=32) introduced into pair housing SP, immunological, and behavioral measures 24 subjects inoculated with SIV SP, viral, immunological, and behavioral measures To aim 4 8: SP antagonist plus Tenofovir after 6 mo. 8: continued treatment with SP antagonist

  23. Aim 4: Compare disease progression in stable and unstable social settings, and compare continuous SP antagonist dosing with intermittent treatment (or periodically increased dose), timed to social rearrangements.Social stress, indices of psychological disturbance, and disease progression • Psychosocial stressors accelerates progression of HIV disease in humans • Social disruption (introductions and separations) accelerates SIV disease progression in macaques • Social disruption alters HPA axis and immune function in primates. • Social stress cannot be experimentally manipulated in humans. • The efficacy of targeting SP antagonists to periods of heightened stress represent an elaboration to the macaque model toward clinical trials in humans.

  24. Aim 4: Compare disease progression in stable and unstable social settings, and compare continuous SP antagonist dosing with intermittent treatment (or periodically increased dose), timed to social rearrangements. All subjects (N=24**), single housing SP, immunological, and behavioral measures * All subjects (N=24) introduced into pair housing SP, immunological, and behavioral measures * All subject (N=24) inoculated with SIV, all pairs rearranged every 2 months 8 treated with consistent dose of SP antagonist 8 treated with intermittent or variable dose, timed to social rearrangements 8 Untreated Longitudinal SP, viral, immunological, and behavioral measures *These data will serve as the baseline data for subsequent within-animal paired comparisons following infection and/or treatment with SP antagonist, as well as contributing data on normal untreated animals **Eight of these animals (SIV-neg controls treated with SP antagonist) will be from aim 3 after a washout

  25. Project 1 and 2 Selection of SP antagonist Relevant cell types to examine Antiviral and immunomodulatory effects Project 4 Objectives of projects 3 and 4 are complementary Core B Evaluate SP antagonists for effects on macaque cells and SIV In vitro studies will inform dose for macaque studies Core C Pharmacokinetics Statistical analysis Study design Project 1 and 2 In vivo relevance of in vitro data Project 4 Safety Long term virologic and immunologic effects Survival Guide future clinical studies Interaction/synergy between Project 3 and other projects and cores Project 3

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