Advances in HIV therapeutics and cure strategies: findings obtained through non-human primate studies.

Human immunodeficiency virus (HIV), the main contributor of the ongoing AIDS epidemic, remains one of the most challenging and complex viruses to target and eradicate due to frequent genome mutation and immune evasion. Despite the development of potent antiretroviral therapies, HIV remains an incurable infection as the virus persists in latent reservoirs throughout the body. To innovate a safe and effective cure strategy for HIV in humans, animal models are needed to better understand viral proliferation, disease progression, and therapeutic response. Nonhuman primates infected with simian immunodeficiency virus (SIV) provide an ideal model to study HIV infection and pathogenesis as they are closely related to humans genetically and express phenotypically similar immune systems. Examining the clinical outcomes of novel treatment strategies within nonhuman primates facilitates our understanding of HIV latency and advances the development of a true cure to HIV.

Transcriptional signatures of Zika virus infection in astrocytes.

Astrocytes are an early and important target of Zika virus (ZIKV) infection in the developing brain, but the impacts of infection on astrocyte function remain controversial. Given that nonhuman primate (NHP) models of ZIKV infection replicate aspects of neurologic disease seen in human infections, we cultured primary astrocytes from the brain tissue of infant rhesus macaques and then infected the cells with Asian or African lineage ZIKV to identify transcriptional patterns associated with infection in these cells. The African lineage virus appeared to have greater infectivity and promote stronger antiviral signaling, but infection by either strain ultimately produced typical virus response patterns. Both viruses induced hypoxic stress, but the Asian lineage strain additionally had an effect on metabolic and lipid biosynthesis pathways. Together, these findings describe an NHP astrocyte model that may be used to assess transcriptional signatures following ZIKV infection.

Adverse event following live attenuated chikungunya vaccine in a cynomolgus macaque with pre-existing chronic hydrocephalus.

A cynomolgus macaque (Macaca fascicularis) with a pre-existing, undiagnosed, subclinical but severe cerebral hydrocephalus was enrolled in a study of long-term immunogenicity of the IRES/CHIK vaccine. The animal began showing signs of neurological dysfunction post-vaccination, which progressed and ultimately resulted in euthanasia. The underlying brain abnormality was revealed at necropsy and was subsequently investigated with gross and microscopic examination. This becomes the first reported case of an adverse event following administration of a live attenuated vaccine and suggests the possibility of an increased susceptibility risk of unwanted adverse outcome associated with vaccination in populations with pre-existing conditions such as hydrocephalus.

The flavivirus dengue induces hypertrophy of white matter astrocytes.

Flaviviruses, including Zika and dengue (DENV), pose a serious global threat to human health. Of the 50+ million humans infected with DENV annually, approximately 1-3 % progress to severe disease manifestations, dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Several factors are suspected to mediate the course of infection and pathogenesis of DENV infection. DHF and DSS are associated with vascular leakage and neurological sequelae. Our hypothesis was that altered astrocyte activation and morphology would alter the dynamics of the extracellular space and hence, neuronal and vascular function. We investigated the mechanisms of neuropathogenesis DENV infection in rhesus macaques. There were decreased numbers of GFAP immunopositive astrocytes per unit area, although those that remained had increased arbor length and complexity. This was combined with structural hypertrophy of white matter astrocytes in the absence of increased vascular leakage. Combined, these studies show how even low-grade infection with DENV induces measurable changes within the parenchyma of infected individuals.

Neuropathogenesis of Chikungunya infection: astrogliosis and innate immune activation.

Chikungunya, "that which bends up" in the Makonde dialect, is an emerging global health threat, with increasing incidence of neurological complications. Until 2013, Chikungunya infection had been largely restricted to East Africa and the Indian Ocean, with cases within the USA reported to be from foreign travel. However, in 2014, over 1 million suspected cases were reported in the Americas, and a recently infected human could serve as an unwitting reservoir for the virus resulting in an epidemic in the continental USA. Chikungunya infection is increasingly being associated with neurological sequelae. In this study, we sought to understand the role of astrocytes in the neuropathogenesis of Chikungunya infection. Even after virus has been cleared form the circulation, astrocytes were activated with regard to TLR2 expression. In addition, white matter astrocytes were hypertrophic, with increased arbor volume in gray matter astrocytes. Combined, these would alter the number and distribution of synapses that each astrocyte would be capable of forming. These results provide the first evidence that Chikungunya infection induces morphometric and innate immune activation of astrocytes in vivo. Perturbed glia-neuron signaling could be a major driving factor in the development of Chikungunya-associated neuropathology.

Glial cell morphological and density changes through the lifespan of rhesus macaques.

How aging impacts the central nervous system (CNS) is an area of intense interest. Glial morphology is known to affect neuronal and immune function as well as metabolic and homeostatic balance. Activation of glia, both astrocytes and microglia, occurs at several stages during development and aging. The present study analyzed changes in glial morphology and density through the entire lifespan of rhesus macaques, which are physiologically and anatomically similar to humans. We observed apparent increases in gray matter astrocytic process length and process complexity as rhesus macaques matured from juveniles through adulthood. These changes were not attributed to cell enlargement because they were not accompanied by proportional changes in soma or process volume. There was a decrease in white matter microglial process length as rhesus macaques aged. Aging was shown to have a significant effect on gray matter microglial density, with a significant increase in aged macaques compared with adults. Overall, we observed significant changes in glial morphology as macaques age indicative of astrocytic activation with subsequent increase in microglial density in aged macaques.

Innate immune activation in the pathogenesis of a murine model of globoid cell leukodystrophy.

Globoid cell leukodystrophy is a lysosomal storage disease characterized by the loss of galactocerebrosidase. Galactocerebrosidase loss leads to the accumulation of psychosine and subsequent oligodendrocyte cell death, demyelination, macrophage recruitment, and astroglial activation and proliferation. To date, no studies have elucidated the mechanism of glial cell activation and cytokine and chemokine up-regulation and release. We explored a novel explanation for the development of the pathological changes in the early stages of globoid cell leukodystrophy associated with toll-like receptor (TLR) 2 up-regulation in the hindbrain and cerebellum as a response to dying oligodendrocytes. TLR2 up-regulation on microglia/macrophages coincided with morphological changes consistent with activation at 2 and 3 weeks of age. TLR2 up-regulation on activated microglia/macrophages resulted in astrocyte activation and marked up-regulation of cytokines/chemokines. Because oligodendrocyte cell death is an important feature of globoid cell leukodystrophy, we tested the ability of TLR2 reporter cells to respond to oligodendrocyte cell death. These reporter cells responded in vitro to medium conditioned by psychosine-treated oligodendrocytes, indicating the likelihood that oligodendrocytes release a TLR2 ligand during apoptosis. TLRs are a member of the innate immune system and initiate immune and inflammatory events; therefore, the identification of TLR2 as a potential driver in the activation of central nervous system glial activity in globoid cell leukodystrophy may provide important insight into its pathogenesis.

Naltrexone treatment reverses astrocyte atrophy and immune dysfunction in self-harming macaques.

The role of glia in the development and treatment of behavioral abnormalities is understudied. Recent reports have observed glial activation in several disorders, including depression, autism spectrum disorders and self-injurious behaviors (SIB). In the current study, we examined SIB in the physiologically and anatomically relevant nonhuman primate (NHP) model. At the Tulane National Primate Research Center (TNPRC), approximately 5% of singly housed macaques develop symptoms of SIB. We have previously demonstrated that naltrexone hydrochloride can be effective in reducing SIB. We have also demonstrated that the astrocytes of animals with SIB are distinctly atrophic and display heightened innate immune activation compared with control animals. We have added a third group of animals (five macaques identified with SIB and treated with oral naltrexone at a dose of 3.2mg/kg) to the previous cohort (six macaques with a history of SIB but not treated, and nine animals with no history of SIB) for this study. Gray and white matter astrocytes from frontal cortical tissue were examined following necropsy. Innate immune activation of astrocytes, which was increased in SIB animals, was markedly decreased in animals receiving naltrexone, as was atrophy of both grey and white matter astrocytes. This was concomitant with improved behavioral correlates. Preventing astrocyte activation in select areas of the brain to reduce injurious behavior is an innovative concept with implications for mental health studies. Differences in multiple areas of primate brain would help determine how self-injurious behavior develops. These studies suggest a stronger role for astrocytes in the cellular events associated with self-injurious behaviors.

Form follows function: astrocyte morphology and immune dysfunction in SIV neuroAIDS.

Cortical function is disrupted in neuroinflammatory disorders, including HIV-associated neurocognitive disorders (HAND). Astrocyte dysfunction includes retraction of foot processes from the blood-brain barrier and decreased removal of neurotransmitters from synaptic clefts. Mechanisms of astrocyte activation, including innate immune function and the fine neuroanatomy of astrocytes, however, remain to be investigated. We quantified the number of glial fibrillary acidic protein (GFAP)-labeled astrocytes per square millimeter and the proportion of astrocytes immunopositive for Toll-like receptor 2 (TLR2) to examine innate immune activation in astrocytes. We also performed detailed morphometric analyses of gray and white matter astrocytes in the frontal and parietal lobes of rhesus macaques infected with simian immunodeficiency virus (SIV), both with and without encephalitis, an established model of AIDS neuropathogenesis. Protoplasmic astrocytes (gray matter) and fibrous astrocytes (deep white matter) were imaged, and morphometric features were analyzed using Neurolucida. Gray matter and white matter astrocytes showed no change in cell body size in animals infected with SIV regardless of encephalitic status. In SIV-infected macaques, both gray and white matter astrocytes had shorter, less ramified processes, resulting in decreased cell arbor compared with controls. SIV-infected macaques with encephalitis showed decreases in arbor length in white matter astrocytes and reduced complexity in gray matter astrocytes compared to controls. These results provide the first evidence that innate immune activation of astrocytes is linked to altered cortical astrocyte morphology in SIV/HIV infection. Here, we demonstrate that astrocyte remodeling is correlated with infection. Perturbed neuron-glia signaling may be a driving factor in the development of HAND.

A novel real-time CTL assay to measure designer T-cell function against HIV Env(+) cells.

BACKGROUND: To increase the immunosurveillance in HIV infection, we used retroviral vectors expressing CD4-chimeric antigen receptors (CARs) to genetically modify autologous T cells and redirect CTL toward HIV. The CD4 extracellular domain targets envelope and the intracellular signaling domains activate T cells. The maC46 fusion inhibitor binds HIV and blocks viral replication. METHODS: We stimulated rhesus PBMCs with antibodies to CD3/CD28 and cotransduced T cells with CD4-CAR and maC46 vectors. CD4-CAR-transduced T cells were added to Env(+) 293T cells at E:T of 1:1. Killing of target cells was measured as reduced impedance. RESULTS: We observed gene expression in 60-70% of rhesus CD3(+) CD8(+) T cells with the individual vectors and in 35% of the cells with both vectors. CD4-CAR-transduced populations specifically killed Env(+) cells. CONCLUSIONS: In these studies, we showed that designer T cells were redirected to kill Env(+) cells. Control of viremia without HAART would revolutionize treatment for HIV patients.

Astrocyte expression of aging-associated markers positively correlates with neurodegeneration in the frontal lobe of the rhesus macaque brain.

Introduction: As the population over the age of 65 increases, rates of neurodegenerative disorders and dementias will rise - necessitating further research into the cellular and molecular mechanisms that contribute to brain aging. With the critical importance of astrocytes to neuronal health and functioning, we hypothesized that alterations in astrocyte expression of aging-associated markers p16INK4a (p16) and sirtuin 1 (SIRT1) with age would correlate with increased rates of neurodegeneration, as measured by FluoroJade C (FJC) staining. Methods: To test this hypothesis, 19 rhesus macaques at the Tulane National Primate Research Center were selected based on the following criteria: archival FFPE CNS tissue available to use, no noted neuropathology, and an age range of 5-30 years. Tissues were cut at 5 µm and stained for GFAP, p16, SIRT1, and FJC, followed by whole-slide imaging and HALO® image analysis for percentage of marker-positive cells and relative intensity of each stain. Results: We found the percentage of p16+ cells increases with age in total cells and astrocytes of the frontal (p = 0.0021, p = 0.0012 respectively) and temporal (p = 0.0226, p = 0.0203 respectively) lobes, as well as the relative intensity of p16 staining (frontal lobe: p = 0.0060; temporal lobe: p = 0.0269). For SIRT1, we found no correlation with age except for an increase in the relative intensity of SIRT1 in the temporal lobe (p = 0.0033). There was an increase in neurodegeneration, as measured by the percentage of FJC+ cells in the frontal lobe with age (p = 0.0057), as well as in the relative intensity of FJC staining in the frontal (p = 0.0030) and parietal (p = 0.0481) lobes. Importantly, increased p16 and SIRT1 expression in astrocytes correlated with increasing neurodegeneration in the frontal lobe (p = 0.0009, p = 0.0095 respectively). Discussion: Together, these data suggest that age-associated alterations in astrocytes contribute to neurodegeneration and provide a target for mechanistic studies in the future.

Transient acidification and subsequent proinflammatory cytokine stimulation of astrocytes induce distinct activation phenotypes.

The foot processes of astrocytes cover over 60% of the surface of brain microvascular endothelial cells, regulating tight junction integrity. Retraction of astrocyte foot processes has been postulated to be a key mechanism in pathology. Therefore, movement of an astrocyte in response to a proinflammatory cytokine or even limited retraction of processes would result in leaky junctions between endothelial cells. Astrocytes lie at the gateway to the CNS and are instrumental in controlling leukocyte entry. Cultured astrocytes typically have a polygonal morphology until stimulated. We hypothesized that cultured astrocytes which were induced to stellate would have an activated phenotype compared with polygonal cells. We investigated the activation of astrocytes derived from adult macaques to the cytokine TNF-α under resting and stellated conditions by four parameters: morphology, intermediate filament expression, adhesion, and cytokine secretion. Astrocytes were stellated following transient acidification; resulting in increased expression of GFAP and vimentin. Stellation was accompanied by decreased adhesion that could be recovered with proinflammatory cytokine treatment. Surprisingly, there was decreased secretion of proinflammatory cytokines by stellated astrocytes compared with polygonal cells. These results suggest that astrocytes are capable of multiple phenotypes depending on the stimulus and the order stimuli are applied.

Cytokines and chemokines at the crossroads of neuroinflammation, neurodegeneration, and neuropathic pain.

Cytokines and chemokines are proteins that coordinate the immune response throughout the body. The dysregulation of cytokines and chemokines is a central feature in the development of neuroinflammation, neurodegeneration, and demyelination both in the central and peripheral nervous systems and in conditions of neuropathic pain. Pathological states within the nervous system can lead to activation of microglia. The latter may mediate neuronal and glial cell injury and death through production of proinflammatory factors such as cytokines and chemokines. These then help to mobilize the adaptive immune response. Although inflammation may induce beneficial effects such as pathogen clearance and phagocytosis of apoptotic cells, uncontrolled inflammation can result in detrimental outcomes via the production of neurotoxic factors that exacerbate neurodegenerative pathology. In states of prolonged inflammation, continual activation and recruitment of effector cells can establish a feedback loop that perpetuates inflammation and ultimately results in neuronal injury. A critical balance between repair and proinflammatory factors determines the outcome of a neurodegenerative process. This review will focus on how cytokines and chemokines affect neuroinflammation and disease pathogenesis in bacterial meningitis and brain abscesses, Lyme neuroborreliosis, human immunodeficiency virus encephalitis, and neuropathic pain.

Microglia activation by SIV-infected macrophages: alterations in morphology and cytokine secretion.

HIV infection in the brain and the resultant encephalitis affect approximately one third of individuals infected with HIV, regardless of treatment with antiretroviral drugs. Microglia are the resident phagocytic cell type in the brain, serving as a "first responder" to neuroinvasion by pathogens. The early events of the microglial response to productively infected monocyte/macrophages entering the brain can best be investigated using in vitro techniques. We hypothesized that activation of microglia would be specific to the presence of simian immunodeficiency virus (SIV)-infected macrophages as opposed to responses to macrophages in general. Purified microglia were grown and stimulated with control or SIV-infected macrophages. After 6 h, aliquots of the supernatant were analyzed for 23 cytokines using Millipore nonhuman primate-specific kit. In parallel experiments, morphologic changes and cytokine expression by individual microglia were examined by immunofluorescence. Surprisingly, the presence of macrophages was more important to the microglial response rather than whether the macrophages were infected with SIV. None of the cytokines examined were unique to co-incubation with SIV-infected macrophages compared with control macrophages, or their supernatants. Media from SIV-infected macrophages, however, did induce secretion of higher levels of IL-6 and IL-8 than the other treatments. As resident macrophages in the brain, microglia would be expected to have a strong response to infiltrate innate immune cells such as monocyte/macrophages. This response is triggered by incubation with macrophages, irrespective of whether or not they are infected with SIV, indicating a rapid, generalized immune response when infiltrating macrophages entering the brain.

Imaging of surface microdomains on individual extracellular vesicles in 3-D.

Extracellular vesicles (EVs) are secreted from all cell types and are intimately involved in tissue homeostasis. They are being explored as vaccine and gene therapy platforms, as well as potential biomarkers. As their size is below the diffraction limit of light microscopy, direct visualizations have been daunting and single-particle studies under physiological conditions have been hampered. Here, direct stochastic optical reconstruction microscopy (dSTORM) was employed to visualize EVs in three-dimensions and to localize molecule clusters such as the tetraspanins CD81 and CD9 on the surface of individual EVs. These studies demonstrate the existence of membrane microdomains on EVs. These were confirmed by Cryo-EM. Individual particle visualization provided insights into the heterogeneity, structure, and complexity of EVs not previously appreciated.

MCP-3/CCL7 production by astrocytes: implications for SIV neuroinvasion and AIDS encephalitis.

Monocyte/macrophages and activated lymphocytes traffic through normal brain, and this trafficking is increased in inflammatory conditions such as HIV encephalitis (HIVE). HIVE is characterized in part by perivascular accumulations of macrophages. The earliest events in this process are poorly understood and difficult or impossible to address in humans. The SIV-infected macaque model of neuroAIDS has demonstrated migration of monocytes into the brain early in disease, coincident with peak SIV viremia. The chemotactic signals that initiate the increased emigration of mononuclear cells into the CNS have not been described. Here, we describe astrocytes as a primary source of chemokines to facilitate basal levels of monocyte trafficking to CNS and that increased chemokine (C-C motif) ligand 7 (CCL7) production may be responsible for initiating the increased trafficking in neuroAIDS. We have previously published complementary in vivo work demonstrating the presence of monocyte chemoattractant protein 3 (MCP-3)/CCL7 within the brain of SIV-infected macaques. Here, we demonstrate that MCP-3/CCL7 is a significant chemokine produced by astrocytes, that basal monocyte migration may be facilitated by astrocyte-derived CCL7, that production of CCL7 is rapidly increased by TNF-α and thus likely plays a critical role in initiating neuroinvasion by SIV/HIV.

Extracellular Vesicles as a Means of Viral Immune Evasion, CNS Invasion, and Glia-Induced Neurodegeneration.

Extracellular vesicles (EVs) are small, membrane-bound vesicles released by cells as a means of intercellular communication. EVs transfer proteins, nucleic acids, and other biologically relevant molecules from one cell to another. In the context of viral infections, EVs can also contain viruses, viral proteins, and viral nucleic acids. While there is some evidence that the inclusion of viral components within EVs may be part of the host defense, much of the research in this field supports a pro-viral role for EVs. Packaging of viruses within EVs has repeatedly been shown to protect viruses from antibody neutralization while also allowing for their integration into cells otherwise impervious to the virus. EVs also bidirectionally cross the blood-brain barrier (BBB), providing a potential route for peripheral viruses to enter the brain while exiting EVs may serve as valuable biomarkers of neurological disease burden. Within the brain, EVs can alter glial activity, increase neuroinflammation, and induce neurotoxicity. The purpose of this mini-review is to summarize research related to viral manipulation of EV-mediated intercellular communication and how such manipulation may lead to infection of the central nervous system, chronic neuroinflammation, and neurodegeneration.

CRISPR based editing of SIV proviral DNA in ART treated non-human primates.

Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues. These proof-of-concept observations offer a promising step toward the elimination of HIV reservoirs in the clinic.

Acquired immunodeficiency syndrome and the blood-brain barrier.

The blood-brain barrier (BBB) plays a critical role in normal physiology of the central nervous system by regulating what reaches the brain from the periphery. The BBB also plays a major role in neurologic disease including neuropathologic sequelae associated with infection by human immunodeficiency virus (HIV) in humans and the closely related simian immunodeficiency virus (SIV) in macaques. In this review, we provide an overview of the function, structure, and components of the BBB, followed by a more detailed discussion of the subcellular structures and regulation of the tight junction. We then discuss the ways in which HIV/SIV affects the BBB, largely through infection of monocytes/macrophages, and how infected macrophages crossing the BBB ultimately results in breakdown of the barrier.

Association of FAK activation with lentivirus-induced disruption of blood-brain barrier tight junction-associated ZO-1 protein organization.

Expression of tight junction proteins between brain microvascular endothelial cells (BMECs) of the blood-brain barrier (BBB) is lost during development of human immunodeficiency virus (HIV) encephalitis (HIVE). Although many studies have focused on the strains of virus that induce neurological sequelae or on the macrophages/microglia that are associated with development of encephalitis, the molecular signaling pathways within the BMECs involved have yet to be resolved. We have previously shown that there is activation and disruption of an in vitro BBB model using lentivirus-infected CEMx174 cells. We and others have shown similar disruption in vivo. Therefore, it was of interest to determine if the presence of infected cells could disrupt intact cerebral microvessels immediately ex vivo, and if so, which signaling pathways were involved. The present data demonstrate that disruption of tight junctions between BMECs is mediated through activation of focal adhesion kinase (FAK) by phosphorylation at TYR-397. Inhibition of FAK activation is sufficient to prevent tight junction disruption. Thus, it may be possible to inhibit the development of HIVE by using inhibitors of FAK.