Project 3

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

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

HypothesisSP 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.

Specific AimsDetermine 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.

Aim 1Determine the cellular distribution of SP and its receptor in tissues of normal and SIV-infected macaques at various stages of disease. Existing data on the distribution of SP and its receptor in nonhuman primates is primarily limited to neurons. The focus of this aim will be on cells of the immune system, particularly monocyte/macrophages in the CNS.Mechanisms of SP and its antagonists in AIDS pathogenesis can not be discerned unless the relevant cell types are defined in vivo.The study will be done using archival tissues collected from SIV-infected macaques and matched controls.

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 macaques8 acute infection + 8 matched controls8 clinically asymptomatic/viral set point + 8 matched controls8 terminal AIDS + 8 matched controlsExamination 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.Detection of SP+ cells by IHC and ISHDetection of NK1R+ cells by IHC and ISHMultiparameter confocal microscopy for cell type and virus (for SIV-infected animals)SPNK1RMacrophageSPNK1RNK cellSPNK1RSIVSPNK1RT cell

Aim 2Characterize 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.

Aim 3Examine the effects of SP antagonists in normal and SIV-infected macaquesEvaluation 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.

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 housingAll subjects (N=32) introduced into pair housingSP, immunological, and behavioral measures *24 subjects inoculated with SIVLongitudinal 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

Aim 3a: Evaluation of the pharmacokinetics of SP antagonists

8 normal uninfected macaques*8 SIV-infected macaques(released from other studies)Collect blood to measure -Antagonist-Viral load (for infected animals)-Serum chemistriesTreat with SP antagonistsTreat with SP antagonists* will utilize 8 of the animals shown used in aims 2/3 either prior to infection or from the uninfected control group

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 housingAll subjects (N=32) introduced into pair housingSP, immunological, and behavioral measures *24 subjects inoculated with SIVLongitudinal 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

8 SIV-pos. subjectsTreated with SP antagonist8 SIV-pos. subjectsTreated with SP antagonist plus tenofovirAim 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.Longitudinal SP, viral, immunological, and behavioral measures16 SIV-pos. subjectsTreated with SP antagonist

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

Preinoculation (weeks)

Post inoculation (weeks)

Postioculation (months)

-18 to -12

-12 to -6

-6 to 0

-2

0

1

2

3

4

Monthly X6

every other month. thereafter

Terminal

Physical exam

weight*

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Blood draw

1) CBC

2) chemistry

3) CD4 count

4) Viral load

5) SIV immune response

6) NK cells

7) SP

8) Cortisol

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CSF tap

SP and SIV

X

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Inoculation with SIV

X

Behavioral data

1 h/wk

1 h/wk

3 h

3 h

3 h

3 h

1 h/wk

1 h/wk

Social introduction

Stable social setting

Unstable social setting

X

X

Mo. 2,4, & 6 to death

Telemetry

Continuous throughout study

Intestinal lymphocytesPeripheral blood lymphocytesMamu-A*01/p11C, C-M7 days p.i.14 days p.i.21 days p.i.63 days p.i. mm352 mm353 mm356mm352 mm353 mm3561.8%4.6%20%5.3%1.3%1.8%11.3%8.5%0.2%3.1%0.1%0.1%0.0%4.1%0.0%17.3%10.5%20%3.4%0.0%11.4%3.0%4.5%8.7%14.7%22.3%Kinetics of SIV-specific CTL responses in early SIV infectionGated through CD3+CD8HI lymphocytes

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

Elevations in SP in humans infected with HIV and macaques infected with SIV

LU20/107PBMCDecreased NK cell activity during SIV infection

Specific AimsDetermine 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.

The macaque model is ideally suited to this projectThe 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.

Neurobehavioral measuresTelemetry data: circadian rhythms, sleep, activity levelBehavioral observation:Species-appropriate social and non-social behavior Abnormal behaviorsPsychological indices of disturbance

Primate models of psychological disturbance: Depressive behaviorsOperational DefinitionSlumped or collapsed body posturePeriods of unresponsivity to environmental stimuliPeriods of hypervigilanceEthopharmacologic validationAmelioration associated with tricyclics and MAO-I inhibitsExacerbated with reserpineConstruct validation (causes and physiological correlates)Associated with social stress as in humansAssociated with altered HPA and dopaminergic function as in humansFace validity: Observational characteristics in humansSlumped or collapsed body postureUnresponsivity to environmental stimuli

Primate models of psychological disturbance: Anxiety-related behaviorsOperational DefinitionSelf-directed displacement behaviorsScratchingSelf-groomingBody shakingEthopharmacologic validationAmelioration (dose dependent) with lorazepam, midazolam, diazepam, clonidineExacerbated with axiogenics (FG142, b-CCE) Construct validation (causes and physiological correlates)Associated with uncertainty and anticipation of aversive conditions as in humansAssociated with autonomic hyperarousal as in humansFace validity: Observational characteristics in humansSelf-directed displacement behaviorsScratchingSelf-grooming

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 housingAll subjects (N=32) introduced into pair housingSP, immunological, and behavioral measures 24 subjects inoculated with SIVSP, viral, immunological, and behavioral measures8: SP antagonist plusTenofovir after 6 mo.8: continued treatment with SP antagonistTo aim 4

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 progressionPsychosocial stressors accelerates progression of HIV disease in humansSocial disruption (introductions and separations) accelerates SIV disease progression in macaquesSocial 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.

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 housingSP, immunological, and behavioral measures *All subjects (N=24) introduced into pair housingSP, immunological, and behavioral measures *All subject (N=24) inoculated with SIV, all pairs rearranged every 2 months8 UntreatedLongitudinal 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 washout8 treated with consistent dose of SP antagonist8 treated with intermittent or variable dose, timed to social rearrangements

Interaction/synergy between Project 3 and other projects and coresProject 1 and 2Selection of SP antagonistRelevant cell types to examineAntiviral and immunomodulatory effectsProject 4Objectives of projects 3 and 4 are complementaryCore BEvaluate SP antagonists for effects on macaque cells and SIVIn vitro studies will inform dose for macaque studiesCore CPharmacokineticsStatistical analysisStudy designProject 1 and 2In vivo relevance of in vitro dataProject 4Safety Long term virologic and immunologic effectsSurvival Guide future clinical studiesProject 3

SIV disease course

Cytokines

IFNgCD3CD68Intracellular cytokine staining to examine immune function in situ

ConclusionsNeuroinvasion by pathogenic SIV occurs within 14 days of infectionPeak vireimiaUpregulation of endothelial adhesion molecules, chemokines and cytokinesPrimary cell type productively infected is the perivascular macrophage (subsets?)Virus specific CTLs appear in the CNS coincident with declines of viral load in the CNSNeuroinvasion is associated with evidence of neuronal dysfunction

P = .014LU20/107 PBMC0100200300400500SIV (n = 3)SIV+ (n = 8)

HIV, Substance P, and Psychological DisturbanceHIV is neurovirulentImpairs fine motor control, memory, emotional control. Causes motor slowing, sleep disturbanceResults in dementia in 25% of casesAssociated with high rates of depressive and anxiety symptomsSP plays a role in depression and anxietyRodent model demonstrates relationship between exogenous SP, stress paradigms, and SP levelsHumans elevated SP associated with the expression of depressive and anxiety-related behaviors; NK1 antagonists ameliorate these symptoms

The SIV/macaque model of neuroAIDSNeuropathology very similar to that seen in HIV-infected humans 25% to 50% of SIV-infected macaques develop SIVE. Greater incidence in rapid progressorsSIV is present in the lesions, primarily within cells of monocyte/macrophage lineageNeuroinvasion occurs within 7 to 14 days of infection coincident with peak viremiaIncreased numbers of perivascular macrophagesEarly changes associated with SIV infection include altered circadian rhythms in body temperature and motor activityAll pathogenic strains of SIV are neuroinvasiveDevelopment of macrophage-tropism is necessary but not sufficient for development of SIVE

Aim 2: Characterize the natural history of SP in normal and SIV-infected rhesus macaques in relationship to viral, immunological, circadian, and behavior measures Aim 3: Examine the effect of SP antagonists in normal and SIV-infected macaques All subjects (N=32), single housingSP, immunological, and behavioral measures *All subjects (N=32) introduced into pair housingSP, immunological, and behavioral measures *24 subjects inoculated with SIV8 SIV-neg. subjectsTreated with SP antagonistLongitudinal 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 2Aim 3b and 3cAim 2To aim 4

My lab has been focused on examining early events in the pathogenesis of AIDS using the SIVmacaque model. Today I will talk about that data in the context of neuroAIDS.Andrew has just outlined how we will track viral and immunological variables to identify how the course of SIV is affected by an SP antagonist, both alone and with concurrent treatment with an antiviral. Im going to go explain how we are going to use behavioral research to further characterize disease course and the effect of treatment. Behavioral will be followed in Aims 2 and 3, which Andrew has already taken you through, as well as Aim 4, which Ill be telling you about.

This proposal brings together three areas of research together to comprise a uniquely suited model for assessing the effects of SP antagonists.The rhesus macaque is the premier animal model for evaluating the antiviral and immunomodilatory effects of compounds used to combat the course of AIDS.The goal of this project is not just to look at these effects, but to study how some of the behavioral effects of HIV, namely depression and anxiety, can be moderated with this same compound.Happily, the macaque has also has a long history of being employed to model psychopathology and is therefore ideally suited for this concurrent evaluation of the physiological and psychological effects of SP antagonists. Like humans, rhesus macaques live in complex societies with persistent social bonds. Dependence on these social bonds means that their manipulalation, including disturbed rearing, social separation, and chronic social instability or subordinance creates social stressors that result in psychopathology that is analagous to psychological problems in humans. The manipulation of social variables and levels of social stress is a productive variable for studying disease progression in nonhuman primates. For example,its already been demonstrated that varying types of social disruption results in accelerated disease progression and reduced survival among SIV+ macaques.Other examples include cariovascular disease and reproductive function.

Subcutaneous radio telemetry devices will allow us to to document changes in circadian rhythms, including sleep/wake patterns, overall activity level, and body temperature. These variables are known to be affected by SIV progression. These data, in conjunction with measures of viral load and immune parameters, will cue us in onto what periods of time to look at more closely with detailed behavioral observations. We will conduct extensive behavioral videotaping in order to document baseline levels of behaviors as well as throughout the course of SIV progression, and will select periods of time based on the physiological and telemetry data to code the data using an exhaustive ethogram of rhesus macaque behavior.Psychopathology has been well characterized behaviorally, physiologically, and pharmacologically in nonhuman primates. There is ample evidence that these behaviors correspond to depression and anxiety disorders in humans. First, with respect to depressive behaviors:Operational DefinitionSlumped or collapsed body posturePeriods of unresponsivity to environmental stimuliPeriods of hypervigalenceEthopharmacologic validationAmelioration associated with tricyclics and MAO-I inhibitsExacerbated with reserpineConstruct validation (causes and physiological correlates)Associated with social stress as in humansAssociated with altered HPA and dopimanergic function as in humansFace validity: Observational characteristics in humansSlumped or collapsed body postureUnresponsivity to environmental stimuli

Anxiety-related behaviors:Operational DefinitionSelf-directed displacement behaviorsScratchingSelf-groomingBody shakingEthopharmacologic validationAmelioration (dose dependent) with lorazepam, midazolam, diazepam, clonidineExacerbated with axiogenics (FG142, -CCE) Construct validation (causes and physiological correlates)Associated with uncertainty and anticipation of aversive conditions as in humansAssociated with autonomic hyperarousal as in humansFace validity: Observational characteristics in humansSelf-directed displacement behaviorsScratchingSelf-grooming

Let me take you again through our research plan for Aims 2 and 3, and then go on to explain our plan for Aim 4.We plan on studying our subjects in a social setting, consisting of protected contact housing. Single housing would be less productive for this study because the psychological indices of disturbance we plan on studying are best examined in a social setting, and can be elicited in a social setting. However, as long as we need to introduce them into a social setting for study purposes, we will backtrack and get behavioral, physiological, and immunological data in the single caged setting as well. We will document the effect of the actual introduction on SP levels and cortisol as well, which will allow us to look at how SP level responds to social stress in our subjects, provide a fallback set of baseline data in case we end up with a subject that must be housed singly later on in the project, and contribute to informing future research of the influence of housing setting.Once individuals have been introduced in to pair housing, another set of physiological and behavioral data will be collected.Following inoculation, we will follow subjects particularly closely due to the rapid physiological changes that accompany initial SIV infection, followed by a more sedate pace of data collection until sacrifice.So in addition to the 8 untreated subjects, the 8 treated with SP, the 8 treated with SP and then tenofavir, Aim 4: Compare disease progression in stable and unstable social settings, and compare continuous NK1 antagonist dosing with intermittent treatment (or periodically increased dose), timed to social rearrangements. Since we know that social stressors influence the time course of both HIV and SIV, this phase will involve monkeys subject to higher levels of social stress than those described so far. Social group rearrangement is a widely used and reliable paradigm for investigating the effects of stress on behavior, physiology, and disease progression.Subjects will move into new social groups every 2 months. These subjects will derive from the 8 SIV-negative subjects used in aim 3 plus 16 additional animals. Procedures will otherwise be identical.This condition may more closely mimic the periodic stressors implicated in accelerated HIV disease progression in humans. It will allow us to model alternative dosing strategies intermittent medicating or altered dosing tied to stressful events. Our project will interact with other projects and cores in the following ways.Projects 1 and 2 will determine which antagonist we study, guide the relevant cell types to examine, and provide preliminary (?) data as to the anticipated antiviral and immunomodulatory effects we will see in vivo.We have designed out project to be complementary to project 4Core B will evaluate SP antagonists for effect on macaque cells and SIV and in vitro studies will inform dose for our studyCore C will guide our pharmacokinetic analysis as well as provide statistical analysis and study design.

Our project in turn will determine the in vivo relevance of the in vitro data before being studied in humans. For the project involving human subjects, our study will guide dose for antiviral (and behavioral???) effects and guide future clinical studies as well.