Jay Rappaport - US grants

Tbe HIV-1 tat gene product is a transactivator of viml gene expression. Tat promotes transcription from the HIV- I LTR, resulting in increased synthesis of viral MRNA. Several lines of evidence suggest that the Tat activation responsive region (terrned TAR), within the HIV- I LTR, is recognized as a stem-looped nascent RNA. Although Tat has been demonstrated to bind to TAR RNA in vitro, the requirements for Tat binding are not sufficient to account for the multiple sequence and structural requirements for transactivation. Our current model of viml gene activation by Tat involves the interaction of Tat with Tar RNA in combination with one more cehular factor(s). In order to further understand the complex mechanism of HIV gene regulation, we will investigate the protein components (Tat and cellular proteins) which participate in the transactivation pathway using genetic and biochemical approaches. In order to further investigate the interaction of Tat with the TAR sequence, we will take advantage of the difference in specificity between HIV-1 and the more closely related HIV-2 and SIV Tat proteins. Using chimeric HIV-L/SIV and HIV-l/HIV-2 tat constructs in transactivation assays, a domain of Tat responsible for LTR specificity will be identified. Mutagenesis within this region will further define the amino-acid residues which specify TAR recognition. The ability of HIV-1 Tat to recognize a smaller target relative to the HIV-2 and SFV proteins, may permit more promiscuous interactions with host gene regulation and may contiibute to the pathogenesis in AIDS. Tbe Tat transactivation pathway involves the function of one or more cellular TAR RNAbinding proteins. Several groups have idenfified TAR RNA-binding activifies in nuclear extracts. Marciniak et al.(1990) have identified a 68 kDa polypeptide (in HeLa cells) which increases transactivation by Tat in cell-free extracts. Our aim is to identify and characterize TAR RNA-binding polypeptides which function, in concert with Tat, to promote HIV- I gene expression in target cells. We have identified a 46 kDa polypeptide in H9 nuclear extracts which recognizes TAR RNA in UV cross-linking assays. We propose to purify this polypeptide and determine its role in HIV- I gene expression. Given the essential role of Tat in HIV-1 viral replication, the Tat activation pathway is an attractive control point for the development of therapeutic moclalities. In addition to increasing our basic understanding of FEV gene regulation, the identification of one or more cellular factor(s) involved in Tat activation would open additional avenues of investigation toward antiviral therapies in AIDS.

DESCRIPTION (adapted from the Abstract): CNS disorders are frequent sequelae of HIV infection. In addition to its direct effect upon infection of a variety of CNS cells, the HIV-1 may also exert its pathogenic effects by indirect mechanisms involving secreted cytokines and immunomodulators. In support of this concept, earlier studies by others and by the Principal Investigator have indicated that the viral regulatory protein, Tat, has the capacity to augment expression of tumor necrosis factor alpha (TNF-alpha), and transforming growth factor beta (TGF-beta) in cells from human and animal brain. The Investigator hypothesizes here that HIV-1 Tat, by de-regulating expression of these important cytokines, may contribute to CNS dysfunction. To examine the hypothesis, the Investigator plans to express Tat and its responsive cytokines, TNF-alpha and TGF-beta, in CNS cells, in vitro and in vivo, and to determine the ability of these proteins to cause alterations which are seen neuro-AIDS. The specific aims are to (1) construct a HSV1-based transduction vector which permits cell type-specific expression of foreign genes in single CNS cell types, and examine the level of its infectivity in a wide range of CNS cells and its fidelity for expressing the foreign gene in the designated CNS cell type in cell culture and animal model systems; (2) utilize the cell type-specific HSV-1 transduction system to express Tat, TNF-alpha, and TGF-beta in single CNS cell types and evaluate the pathological alterations caused by over-expression of these regulatory proteins in brain; (3) develop an inhibitory/therapeutic modality utilizing ribozymes to abrogate expression of Tat, TNF-alpha, and TGF-beta in animals experiencing CNS diseases as the result of over-expression of Tat, TNF-alpha, and TGF-beta, and determine the ability of these therapeutic agents to prevent or improve CNS disease manifestation. As proposed, the results of these studies will provide fundamental and detailed information regarding the role of cytokines induced by HIV-1 Tat in the CNS pathogenesis of AIDS and a wide range of other neurological disorders.

HIV infection induces a spectrum of neurological disorder in at least two thirds of patients with AIDS. In addition to behavioral and motor abnormalities, these manifestations are histologically apparent as multifocal giant cell encephalitis and diffuse white matter degeneration. Although microglia are the major reservoir of HIV in CNS, brain tissue exhibits extensive pathogenic alterations including apoptosis of neurons, reactive astrocytes, cytolytic destruction and demyelination. The pathogenesis of HIV in CNS involves changes in virus infected cells, which are manifested in cytokine dysregulation and release of viral factors. One of the factors which has received considerable attention is the HIV Tat protein. Tat is released from infected cells and can serve as a transactivator of cellular as well as viral promoters. The ability Of Tat to be released and taken up into cells permits this protein to function in a dysregulatory capacity through autocrine and paracrine pathways. Tat activates gene expression not only through its own LTR, but can also activate transcription of other viral promoters, notably the JCV late promoter. JCV, a human papovavirus which is the causative agent of PML, infects oligodendrocytes and its replication is increased in patients with HIV associated PML. Our hypothesis is that HIV Tat protein released from infected microglia activates JCV through a paracrine mechanism. Oligodendrocytes are rarely, if at all, infected with HIV, although we have observed this cell type to contain a significant amount of Tat protein in brain tissue of AIDS patients with PML. In our proposed studies we will develop an in vitro model consisting of co-cultures of HIV infected primary microglia and JCV infected primary oligodendrocytes to investigate the intercommunication between H~V-l-infected microglia and JCV-infected oligodendrocytes via Tat protein. Based on our preliminary studies, activation of both JCV and HIV by Tat requires a cellular protein, Puralpha. Puralpha is an activator of both JCV and MIV transcription and mediates the action of Tat through the formation of a ternary complex consisting of Puralpha, Tat, and RNA. Our proposed studies will investigate the role of Puralpha interaction in MIV and JCV gene expression and replication in primary microglia and oligodendrocytes respectively. These studies will further investigate the mechanism of Puralpha interaction through RNA in order to develop protein based therapeutics which will block the action of Tat in promoting HIV and JCV infection.

Until recently, the majority of vaccine efforts directed against HIV-1 have targeted the structural proteins. These efforts are rooted in the empirical nature of vaccine development and prior successes with other viral pathogens. In this proposal, we provide evidence that an HIV-1 regulatory protein, namely Tat, is an excellent vaccine target based on its immunosuppressive properties. We provide evidence that anti-Tat antibody correlates inversely with p24 antigenemia and furthermore, that anti-Tat antibody is a good prognostic indicator for clinical stability. In addition to the role of Tat as a viral transactivator, HIV-1 Tat primes non-infected T cells for activation induced apoptosis. Tat acts extracellularly to inhibit proliferation of T cells in response to recall antigens and enhances the permissivity of non-infected primary cells to HIV infection. Tat therefore is critical for viral replication and dissemination and plays an important role in viral induced immune suppression. To develop a vaccine against Tat, which can be considered an immune toxin, we have developed methodologies for large scale purification and chemical modification, producing an inactive, yet immunogenic protein, "Tat Toxoid." We have recently demonstrated the safety and potency of this vaccine approach in a Phase I clinical trial in 14 HIV infected individuals. In this "therapeutic" vaccine trial, a statistically significant increase in CD4+ cells was observed along with a trend toward a reduction in viral loads. The ultimate goal of this approach will be to develop effective "protective" vaccine strategies for seronegative individuals. Numerous difficulties are inherent in protective HIV-1 vaccine trials in humans, namely, the large number of volunteer participants required. To reduce this number, patients at high risk for HIV-1 infection can be considered, however, ethical considerations mandate counseling regarding behavioral risks for HIV-1 infection, adding to the complexity of such endeavors. In order to develop an effective protective vaccine strategy suitable for human testing, we believe that animal models for vaccination and virus challenge should be utilized. In order to test the efficacy of Tat Toxoid as a protective vaccine component, we propose to immunize rhesus macaques and challenge these animals with a chimeric human/simian immunodeficiency virus (SHIV) containing the HIV-1 Vpu, Envelope, Tat, and Rev proteins. The SHIV isolate, 89.6P, induces CD4+ cell depletion and AIDS like illness in macaques. Our proposed studies will determine the safety, immunogenicity, and efficacy of the Tat Toxoid vaccination and determine the effect of Tat neutralization in enhancing humor and cellular immunity to other viral antigens (i.e. Envelope). The epitope specificity of anti-Tat antibodies produced in rhesus macaques will be determined and compared to neutralization activity in biologic assays.

Until recently, the majority of vaccine efforts directed against HIV-1 have targeted the structural proteins. These efforts are rooted in the empirical nature of vaccine development and prior successes with other viral pathogens. In this proposal, we provide evidence that an HIV-1 regulatory protein, namely Tat, is an excellent vaccine target based on its immunosuppressive properties. We provide evidence that anti-Tat antibody correlates inversely with p24 antigenemia and furthermore, that anti-Tat antibody is a good prognostic indicator for clinical stability. In addition to the role of Tat as a viral transactivator, HIV-1 Tat primes non-infected T cells for activation induced apoptosis. Tat acts extracellularly to inhibit proliferation of T cells in response to recall antigens and enhances the permissivity of non-infected primary cells to HIV infection. Tat therefore is critical for viral replication and dissemination and plays an important role in viral induced immune suppression. To develop a vaccine against Tat, which can be considered an immune toxin, we have developed methodologies for large scale purification and chemical modification, producing an inactive, yet immunogenic protein, "Tat Toxoid." We have recently demonstrated the safety and potency of this vaccine approach in a Phase I clinical trial in 14 HIV infected individuals. In this "therapeutic" vaccine trial, a statistically significant increase in CD4+ cells was observed along with a trend toward a reduction in viral loads. The ultimate goal of this approach will be to develop effective "protective" vaccine strategies for seronegative individuals. Numerous difficulties are inherent in protective HIV-1 vaccine trials in humans, namely, the large number of volunteer participants required. To reduce this number, patients at high risk for HIV-1 infection can be considered, however, ethical considerations mandate counseling regarding behavioral risks for HIV-1 infection, adding to the complexity of such endeavors. In order to develop an effective protective vaccine strategy suitable for human testing, we believe that animal models for vaccination and virus challenge should be utilized. In order to test the efficacy of Tat Toxoid as a protective vaccine component, we propose to immunize rhesus macaques and challenge these animals with a chimeric human/simian immunodeficiency virus (SHIV) containing the HIV-1 Vpu, Envelope, Tat, and Rev proteins. The SHIV isolate, 89.6P, induces CD4+ cell depletion and AIDS like illness in macaques. Our proposed studies will determine the safety, immunogenicity, and efficacy of the Tat Toxoid vaccination and determine the effect of Tat neutralization in enhancing humor and cellular immunity to other viral antigens (i.e. Envelope). The epitope specificity of anti-Tat antibodies produced in rhesus macaques will be determined and compared to neutralization activity in biologic assays.

DESCRIPTION: (Provided by Applicant) In order to evaluate early proteins as therapeutic vaccine targets in AIDS, we will perform immunization studies in macaques previously infected with SIV. This SIV model most appropriately reflects the pathogenesis of HIV in humans. These studies are designed to extend our work developing a vaccine approach targeting HIV-1 Tat protein. We will initially compare the potency and potential efficacy of an SIV Tat Toxoid vaccine in animals initiating HAART therapy during the chronic phase of SIV infection. Immune responses to early and late viral antigens, including the immunogen, will be monitored prior to HAART, during HAART and after discontinuation of HAART. We will test the hypothesis that the Tat vaccine, alone or in combination with low dose IL-2, can promote anti-viral immune responses in chronically infected animals. Sequence analysis and peptide mapping studies will be used to determine if selective pressure is induced by vaccine administration, to characterize the immune response, and to identify escape variants. A second animal study will be performed during the 3rd and 4th year which will deliver a combination of the early proteins. Tat, Rev, and Nef, where Rev and Nef are fused to the basic domain of Tat for uptake. Our strategy will take advantage of the ability of this region of Tat to enter the MHC class-I processing pathway and elicit CD8+ T cell responses to the fused protein component. This strategy will further test the hypothesis that multiple early gene targets may better control virus replication and minimize escape, particularly in view of the function of Nef in downregulating MHC class-I expression. The comparison of a late gene fused to Tat (i.e. Tag-Gag) will permit the determination of the added contribution of a late gene target where immune responses have been previously well characterized in this model. These studies are designed to evaluate the concept of a therapeutic Tat vaccine component in an animal model for lentiviral infection. Since our protein-based strategy stimulates both humoral and cellular (CD4+ and CD8+) immune responses, we propose to extend this protein-based strategy with additional targets. By analogy with antiviral pharmacotherapy, a multi-target vaccine should provide the best long-term protection from viral escape. The goal of this project is to generate the necessary information in an animal model to determine the feasibility of this approach. This information should provide the conceptual framework for a protein vaccine strategy in humans based on Tat and including additional early viral immunogens. If successful, this strategy may be beneficial in further controlling HIV infection and may permit patients treated with HAART during chronic infection to realize independence from pharmacotherapeutic regimens.

[unreadable] DESCRIPTION (provided by applicant): Neurodegenerative disorders including HIV-associated dementia (HAD) are observed in approximately 20 percent of patients with HIV infection, principally those with AIDS. Recent studies demonstrating the concordance of viral quasi-species in CNS and those in bone marrow and PBMCs, together with the neurocognitive improvements observed with HAART therapy, have suggested an important role for events occurring outside the CNS compartment in the development of HIV associated dementia. In circulation, increased numbers of CD14+/CD16+ positive monocytes have been observed in HIV infected individuals with AIDS, with even higher levels among persons with HAD. The abnormal numbers of these activated cells has been proposed to provide a mechanism for CNS invasion and neuronal injury. Our preliminary studies in CNS tissues from patients with HIV encephalitis (HIVE) demonstrate that CD14+/CD16+ macrophages accumulate in the perivascular space and in "microglial" nodules. These cells appear be derived from the expanded activated monocyte subset in circulation as determined by CD45/LCA staining. In addition to the relatively transient perivascular macrophages (CD 14+/CD 16+), our preliminary studies also identify many CD14-/CD16+ mononuclear phagocytes (MPs) in areas of white matter distant from blood vessels, with a ramified morphology. Both perivascular and parenchymal mononuclear phagocytes (MPs) represent reservoirs of HIV infection in CNS. The large increases in the numbers of total perivascular and parenchymal CD68+ MPs we observed, in the absence of proliferation, support our hypothesis that abnormal monocyte/macrophage/trafficking mechanisms contribute to pathogenesis of HIV in CNS. Furthermore, we have observed increased accumulation of CD 16+ MPs, not only in brain, but in multiple organs in patients with HIVE including spleen, lymph node, kidney, and liver. In kidney, we further observe histologic evidence for underlying HIV associated nephropathy in tissue specimens from patients with HIVE. These results suggest a major role for monocyte/macrophage invasion and accumulation in the pathogenesis of AIDS in CNS as well as other organs. In our proposed studies (Specific Aim I), we will determine the relationship between macrophages in CNS, bone marrow, blood, and visceral tissues in HIVE using immunohistochemical and viral-genetic approaches. These studies will be further extended to an in vivo animal model (Specific Aim II) in SIV infected Rhesus macaques. Here we will investigate the expansion of activated monocyte/macrophages and their invasion into CNS and visceral tissue during the course of infection leading to AIDS and SIV encephalitis. We will further examine the migration of labeled autologous monocyte/macrophages into various organs and determine monocyte turnover kinetics in SIVE, SIV/AIDS, and control animals. By combining viral-genetic and tissue based studies in human specimens and in in vivo non-human primate studies, we will gain important information regarding monocyte/macrophage production, trafficking and abnormal organ accumulation in HIVE. We anticipate that these studies will provide novel concepts regarding the role of abnormal monocyte/macrophage production and trafficking in the pathogenesis of AIDS and HIVE, and provide a rationale for novel treatment strategies targeting the monocyte/macrophage developmental program, activation and/or trafficking. [unreadable] [unreadable] [unreadable] [unreadable]

HIV-1 infection of the central nervous system induces a variety of clinical abnormalities including dementia, ataxia, and memory loss. Progressive multifocal leukoencephalopathy (PML) represents one of the most common neurological complications of HIV-1 infection. PML is a fatal demyelinating disease that results from the reactivation of the human neurotropic polyomavirus, JCV, and its infection of oligodendrocytes and astrocytes. Once a rare disorder, the higher incidence of PML among AIDS patients suggests cross-communication between HIV-1 and JCV in the brain. Results from molecular biology and virology studies have established the ability of Tat to augment the JCV genome. This event requires, at least in part, a cellular protein named Pur-alpha, a single stranded DNA and RNA binding protein whose expression is controlled during brain development. Pur-alpha also stimulates HIV-1 gene expression and its association with Tat augments Tat activation of the LTR. Furthermore, Pur-alpha controls JCV DNA replication and gene expression in glial cells by interacting with the JCV early protein, T-antigen. Pur-alpha has an unusual structural feature allowing the protein to interact with various important cellular proteins in addition to nucleic acids. Ablation of Pur-alpha in animal models causes incomplete brain development. Our preliminary observations have shown the ability of Pur-alpha to control cell cycle progression and prolong cells with damaged DNA in S-phase. On the other hand, Pur-alpha has shown the ability to interact with Rad51 (a key factor in the homologous recombination pathway), decrease the level of Rad51 gene transcription, and interfere with the function of Ku70, one of the major components of the non-homologous end-joining pathway. These observations ascribed a new role for Pur-alpha as a gatekeeper of DNA repair, which ensures the efficient and appropriate repair of DNA with high levels of fidelity and accuracy by modulating cell cycle progression and the level of expression and activity of factors involved in cellular DNA repair machinery. Support for this observation stems from our results showing substantial chromosomal abnormalities associated with dysfunctional repair in cells lacking Pur-alpha. In light of these observations one can envision a model in which the physical interaction of Pur-alpha with the JCV regulatory protein, T-antigen, and the HIV-1 transactivator protein, Tat, will have a functional consequence on the ability of Pur-alpha to execute its role in DNA repair during the course of HIV-1 infection and JCV reactivation. In this project, experiments are proposed to delineate the effect of Pur-alpha on DNA repair mechanisms seen in astrocytes and oligodendrocytes, and determine the impact of JCV and HIV-1 upon Pur-alpha functions in cell cycle regulation and genomic stability. The outcome of these studies will provide important information which can be utilized to better understand the indirect communication of two distinct viruses by cellular proteins and their cooperative role in the progression of diseases in the CNS.

Greater than one third of all HIV infections and AIDS cases are related directly or indirectly to injection drug use [1, 2]. While it is clear that drug use itself plays an important role in the spread of HIV infection, the effects of the drugs on HIV disease progression are not fully understood and various epidemiologic, animal and in vitro studies have provided some conflicting results. In view of the confounding variables in human epidemiologic studies and problems in previous animal studies investigating this issue, additional studies are needed to determine the role of opioids in contributing to the pathogenesis of HIV infection and the development of AIDS associated CMS disorders. The Specific Aim of this core is to carry out Experimental Animal Protocols in Indian rhesus macaques, to perform certain clinical, virologic, and immunologic measurements and to provide a mechanism of distribution of samples from the ongoing animal protocols to projects and Core D (Immunohistochemical Core) for further analysis. The Core will provide housing, care, scheduled drug treatment interventions, sample collections, clinical and virologic data collection for two sequential protocols to determine the effects of opioids on the pathogenesis of SIV infection and the development of CMS disease. The activities of this core are essential to the overall performance of this Program Project especially since Project 1,2,3, and 4 rely heavily on the samples from rhesus macaques in various treatment groups as well as on clinical and virologic data generated in this core. The proposed studies that this Core will support are critical for our understanding of how opioid drug abuse affects the course of HIV disease progression.. The experimental design of the Experimental Animal Protocol should lead to definitive answers regarding the role of opiates drugs of abuse in altering the pathogenesis of HIV/SIV and in contributing to CMS disease. These studies supported by this Core should provide important insights regarding pathogenesis and therapeutic approaches in patients with HIV infection, particularly those who are opiate drug abusers.

[unreadable] DESCRIPTION (provided by applicant): This application provides a plan for an Interdisciplinary and Translational Research Training Program (ITRTP) for predoctoral students in NeuroAIDS and related areas of research. This is a joint program from two institutions, Temple University and Drexel University, located in close proximity in Philadelphia, 'Pennsylvania. This program will also integrate training activities and research resources available in a long- tending M.D./Ph.D. and clinical research training programs in AIDS and Neurovirology based in the Department of Neurology, University of Pennsylvania. The proposed plan will create a city-wide interdisciplinary and translational research training program in NeuroAIDS through shared resources, joint research seminar series, journal club, symposia, invited speakers, thesis mentoring and educational opportunities at both institutions. The graduate curriculum at both institutions is designed to provide a broad based scientific foundation in biomedical science including Neuroscience, Immunology, Microbiology, Pharmacology and Physiology. This curriculum including Scientific Communication, Scientific Integrity and Bioethics, and Statistics, as well as courses in Molecular and Cellular Neurobiology and Pathogenesis of Neurobiological Diseases will prepare graduate student for thesis research in NeuroAIDS. Within Temple University School of Medicine, research opportunities in NeuroAIDS and related areas are available within the Department of Neuroscience, Department of Microbiology and Immunology, Department of Pharmacology, and Department of Physiology. Additional research opportunity is available in the School of Engineering. At Drexel University College of Medicine, research opportunities in NeuroAIDS and related areas are available in the Department of Microbiology and Immunology, Department of Neurobiology and Anatomy, Department of Pharmacology and Physiology and Department of Biochemistry and Molecular Biology. Additional research opportunities are available in the School of Biomedical Engineering and Health Science Systems. This program brings together multiple biomedical basic science departments with biomedical engineering at two institutions, and integrates joint training activities with nearby University of Pennsylvania. With the inclusion of clinical AIDS investigators, the proposed program is not only interdisciplinary, but exposes students to training in neuroAIDS basic sciences to AIDS and NeuroAIDS related clinical perspectives. Investigators at Temple and Drexel have built strong, productive and well-funded NeuroAIDS research programs that are now sufficiently established to support formal comprehensive training in NeuroAIDS in areas ranging from molecular studies, in vitro systems, and pathogenesis studies in human and non-human. [unreadable] [unreadable] [unreadable]

Greater than one third of all HIV infections and AIDS cases are related directly or indirectly to injection drug use [1, 2]. While it is clear that drug use itself plays an important role in the spread of HIV infection, the effects of the drugs on HIV disease progression are not fully understood and various epidemiologic, animal and in vitro studies have provided some conflicting results. In the studies proposed here, we plan to address these issues in the context of SIV infection of rhesus macaques (Macaca mulatta). In this project, we will investigate the role of opioid (morphine) treatment in modulating the progression of HIV/AIDS using the SIVmac251 infected Indian rhesus macaque model. We will further determine if morphine plays a role in promoting NeuroAIDS through increased CNS invasion of SIV infected and/or uninfected macrophages. The proposed studies are critical for our understanding of how opioid drug abuse and replacement therapy affects the course of HIV disease progression. The studies proposed here may lead to new approaches to HIV therapeutics based on the u-opioid receptor agonists or antagonists. The proposed studies will be carried out in the context of two Specific Aims. Specific Aim 1 will determine if opioid treatment modulates the course of HIV/SIV disease progression using an SIV infected Indian rhesus macaque model. Specific Aim 2 will determine the effect of opioids in promoting the development of neuroAIDS in the SIV infected Indian rhesus macaques. The experimental design in these studies should lead to definitive answers regarding the role of opiates drugs of abuse in altering the pathogenesis of HIV/SIV and in contributing to CNS disease. These studies may provide important insights regarding approaches to intervention and therapy in patients with HIV infection, particularly those who are opiate drug abusers.

DESCRIPTION (provided by applicant): HIV associated CNS disorders represent a major health problem in a significant proportion of HIV infected individuals. These disorders include minor cognitive and motor disorders, HIV dementia, as well as peripheral neuropathy. The neurologic complications of AIDS develop, in part, because of the invasion of macrophages derived from peripheral blood into the central and peripheral nervous system tissues. Our previous studies have identified the accumulation of a monocyte subset (CD14+/CD163+/CD16+) in peripheral blood in patients with HIV infection and SIV infected rhesus macaques. These monocytes in circulation appear to represent precursors of the perivascular macrophages accumulating in HIV/SIV encephalitis. Normally low in healthy individuals, this subset appears to expand to a degree correlating with HIV/SIV plasma viral load, and inversely with CD4+ T cell count. Based on the phenotype of these cells, the immunosuppression observed in AIDS, together with new information regarding the phenotype and function of distinct monocyte/macrophages subsets, we propose that CD14+/CD163+/CD16+ monocytes accumulating in AIDS, play a role in immune polarization and may contribute to the immunopathogenesis of AIDS. In the studies proposed in this application, we will utilize the SIV infected rhesus macaque model to study the consequences of SIV infection in altering monocyte/macrophage dynamics using the simultaneous combination of two in vivo cell tracking approaches. An approach utilizing CFSE fluorescent dye and BrdU labeling will be employed to study monocyte/macrophage dynamics in SIV infected, SIV infected/CD8+ T cell depleted, and healthy control animals. In order to identify potential effects solely attributable to the CD8+ T cell depletion and to further understand normal homeostatic processes, uninfected CD8+ T cell depleted animals will also be studied. We will further determine if the CD14+/CD163+/CD16+ monocyte exhibits immune polarization using antigen specific immune assays and the characterization of cytokines, chemokines as well as regulatory pathways using multiplex (Luminex) and Affymetrix gene chip approaches. The studies proposed here may provide new avenues for investigation and the development of therapies targeting the monocyte/macrophage in AIDS and NeuroAIDS. PUBLIC HEALTH RELEVANCE: HIV infection is major problem worldwide. The monocyte/ macrophage system not only provides a stable reservoir for the virus, but these cells are responsible for trafficking of HIV into the CNS and other tissues. The studies proposed here may have a major impact on our understanding of the function of the monocyte/macrophage in the development of CNS disease, and at the same time investigate their role in contributing to immune dysfunction in AIDS.

DEVELOPMENTAL CORE There is an urgent need for advances in neuroAIDS research that may lead to improved diagnostics and patient care. Such advances require an interdisciplinary approach with participation of investigators in basic, behavioral and clinical research. The major goal of the Comprehensive NeuroAIDS Center Cores (CNACC) is to facilitate interaction between basic science, behavioral research and clinical medicine by providing a mechanism of support for translational investigations through infrastructural and financial support, as well as mentorship. Toward this end, the Developmental Core will provide support for pilot investigations, progress evaluation, guidance, and mentorship. This core application describes the experience of the Core Leaders, the process to recruit and review innovative project applications, and the role the Core will play in mentorship of clinical and basic science faculty and trainees at various levels. It is anticipated that this core will play a pivotal role in enhancing and ensuring the success of neuroAIDS investigators, the advancement of neuroAIDS research and patient care, and the CNACC mission.

DESCRIPTION (provided by applicant): HIV infection of the CNS persists despite antiretroviral treatment, representing a challenge for treatment of neurologic disorders and a significant hurdle for the eradication of HIV in treated patients. It is likely that elimination ofHIV infected reservoirs in the brain will require interventions designed to stimulate immune recognition of HIV infected cells and/or circumvent immune suppressive mechanisms. Adenosine triphosphate (ATP) is a potent immune stimulant, acting via P2-type receptors on immune cells. Adenosine, in contrast, has potent immune suppressive activities on T cells and macrophages/dendritic cells via P1-type receptors. Microglia in CNS, similar to an action of regulatory T cells, express ectonucleotidases CD39 and CD73, which convert ATP and ADP to AMP (via CD39), with further metabolism to adenosine (via CD73), providing a potentially important mechanism mediating immune modulation. In our preliminary studies in CNS tissues from SIV infected rhesus macaques, both CD39 and CD73 exhibit increased expression relative to uninfected control brain tissue. The expression of both ectoenzymes is markedly increased on parenchymal microglia in SIV infected brain, even in the absence of encephalitis. In addition, CNS perivascular macrophages and cells within microglial nodules, highly express these enzymes in the setting of SIV encephalitis In our proposed studies, we will test the hypothesis that ATP metabolism, via the enzymatic activities of CD39 and CD73 expression on microglia, is increased in the CNS in SIV infected rhesus macaques, contributes to the survival of infected cells, and impairs virus specific immune responses. In Specific Aim I, we will test the hypothesis that CD39 and CD73 expression on microglia, is increased in SIV infection and determine the impact of cART treatment on expression and enzymatic activity. We will further evaluate ATP metabolites as biomarkers in CSF during the course of this study and determine if values correlate with SIV infection status, biologic and virologic markers for pathogenesis, CD39 and CD73 expression in CNS, and the response to cART treatment. We will test the hypothesis increased CD39 and CD73 expressed on brain derived microglia in SIV infection contributes to ATP metabolism, suppresses T cell responses, and increase survival of microglia in vitro. We will measure and compare ectonucleotidase activities, the ability to suppress autologous SIV specific immune responses, and the sensitivity to ATP induced cell death, using CNS derived microglia from uninfected controls, SIV infected, and SIV infected rhesus macaques treated with cART. We anticipate that our proposed studies will provide a novel avenue for therapeutics to augment immune mediated clearance of HIV and other CNS pathogens.

DESCRIPTION (provided by applicant): HIV infection of the central nervous system (CNS) represents an important challenge for both the treatment of neurocognitive disorders as well as the eradication of HIV infection from CNS reservoirs. The eradication of CNS reservoirs of infection will likely be beneficial, not only for treating cognitive dysfunction, but also for treament of HIV infection overall. It is clear that neurocognitive impairment is a negative predictor of survival in HIV infection, suggesting common mechanisms of viral persistence and/or pathogenesis promote both immune deficiency and neurocognitive impairment. At the intersection of both these disorders is the macrophage, where altered immune polarization can suppress antiviral immune reactions and at the same time, serve as a reservoir for viral infection in the CNS, as well as a source of inflammation and neuronal injury. This proposal is a result of a joint interactions between two NIMH supported NeuroAIDS related Centers (P30s) at Temple University (Comprehensive NeuroAIDS Center, Kamel Khalili, Ph.D., Program Director) and UPenn/Children's Hospital (Penn Mental Health Research Center, Dwight Evans, M.D., Program Director). Discussions between CHOP/UPENN and Temple investigators including Jay Rappaport, Ph.D. (Temple University), Tracy Fischer-Smith, Ph.D. (Temple University), and Steven D. Douglas, M.D., with the further interactions with Mark G. Lewis, Ph.D., Bioqual, have led to the conception of an interdisciplinary and translational program project application, investigating therapeutic approaches to eradicating HIV infected reservoirs in the CNS. The application is a multiple PI submission: Jay Rappaport, Ph.D. (Corresponding PI, Temple University) and Steven D. Douglas, M.D. (PI-Children's Hospital). This program project in response to PAR-13-267 (Novel NeuroAIDS Therapeutics, Integrated Preclinical/Clinical Program (PO1), entitled NeuroAIDS Therapeutics-Targeting Immune Polarization of Macrophages in CNS, brings together three projects and three scientific cores, housed at three institutions. These institutions include Temple University School of Medicine, Philadelphia, PA, (Project1, Project 2, Core A and Core B), Children's Hospital of Philadelphia (Project 3 and Co-PI Core A), Bioqual Inc., Rockville, MD (Core C). There are a number of important converging concepts in each of the Projects within this program, which support the overarching hypothesis put forth in this program, that altered immune polarization of the monocyte/macrophage lineage promotes immune dysfunction and the persistence of HIV infection in the CNS and the periphery. A critical concept is the importance of interaction between the CNS and the periphery in HIV infection leading to CNS disease. These processes are centered, to a large degree, on alterations within the myeloid lineage, including peripheral monocytes, tissue macrophages, and microglia. In order to eradicate reservoirs of HIV infection, we propose the intervention within three pathways that contribute to the immune polarization of the myeloid lineage. These three approaches target the following pathways: 1) ATP hydrolysis, where the end product, adenosine, is immune suppressive (Project 1, Jay Rappaport, Ph.D., Project Leader, Temple University), 2) cFMS signaling, where M-CSF and IL-34 ligands for this receptor may contribute to abnormal monocyte/macrophage homeostasis, immune polarization, and survival of infected reservoirs, (Project 2, Tracy Fischer-Smith, Ph.D., Project Leader, Temple University) and 3) neurokinin-1 receptor signaling, where the ligand substance P appears to promote virus infection, immune polarization, survival, and CNS invasion of monocytes/macrophages and the establishment of CNS inflammatory and persistent reservoirs (Project 3, Steven D. Douglas, M.D., Project Leader, Children's Hospital of Philadelphia/UPENN). In the studies proposed in this highly integrated program project, we investigate therapeutic approaches targeting each of these pathways in studies, in vitro, ex vivo, and finally in SIV infected rhesus macaques, alone, in combination, and in the context of cART therapy. The goals and objectives of these projects are supported by an Administrative Core (Core A; Jay Rappaport, Ph.D. and Douglas, M.D., Core Co-Leaders), an Immunopathology Core (Core B; Tracy Fischer-Smith, Ph.D., Core Leader, Temple University), as well as a Non Human Primate Core (Core C; Mark G. Lewis, Ph.D., Bioqual, Inc.). It is anticipated that successful completion of the objectives of this overll program, will make important contributions toward the treatment of HIV infection and eradication of HIV reservoirs, including the CNS. It is anticipated that advances made here with further inform efforts aimed at preventive and therapeutic vaccines, where post-exposure adjuvant approaches could be employed to reactivate otherwise suppressed immune reactions.

PROJECT 1: SUMMARY HIV infection of the CNS is believed to play an important role in the development of HIV associated neurocognitive disorders (HAND). In our preliminary studies we identified expanded CD163+/CD16+ monoctyes in circulation as well as CD163+/CD16+ macrophages/microglia in CNS in HIV/SIV infection. These cells likely represent an immune polarizing function, contributing to impaired cellular immunity in HIV infection. It is likely that immune polarization of macrophages toward and immune polarized or suppressive phenotype contributes to HIV persistence in the setting of antiretroviral therapy and contributes to the neurocognitive impairment that persists, despite antiviral therapy. In our recent preliminary studies presented here, we identified increased expression of ectonucleotidase enzymes on the surface of macrophages/microglia in CNS in rhesus macaques infected with SIV. We further provide evidence using an inhibitor of CD39 (and ectonucleotidase), showing inhibition of CD16 and CD163 expression on primary monocytes/macrophages. It is likely, therefore, that pharmacologic modulation of ATP hydrolysis can have therapeutic implications for restoring antiviral immune responses in HIV infection. These observations, together with the immunosuppressive and immune polarizing role of these enzymes, suggest that successful eradication of reservoirs of HIV infection in the CNS will require targeted approaches to prevent increased ATP hydrolysis in order to accomplish eradication of reservoirs of HIV infection, particularly in the CNS. Here we propose in vivo and ex vivo using the SIV infected rhesus macaque model in the context of the following Specific Aims: Specfic Aim 1: To test the hypothesis that Adenosine production, as a consequence of ATP hydrolysis, modulates monocyte maturation (i.e. CD16 gene expression), M2 polarization, and SIV specific immune responses in vitro. Here we will determine the ability of antagonists of CD39 (POM-1) and CD73 (APCP) enzyme activity to inhibit CD16 expression on monocytes in response to treatment with Giant Cell Tumor Conditioned Medium or Macrophage Colony-Stimulating Factor (M-CSF), as inducers of M2 maturation and CD16 expression. In our preliminary data we present evidence suggesting the ability of POM- 1 treatment to inhibit the induction of CD16 expression on monocytes in vitro. As this effect is likely mediated by preventing adenosine formation, we will further investigate the ability of adenosine agonists to promote the maturation of non-classical monocyte subsets (CD16+/CD163+) monocytes derived from normal blood donors and uninfected rhesus macaques. Our in vitro studies will determine the ability of CD39, CD73, and adenosine receptor (ie. Adora2b) antagonists to reduce the maturation of CD16+ monocytes in response to M-CSF treatment in vitro, and further, for the ability to reduce CD16 gene expression on cells previously differentiated with M-CSF. The ability of compounds to inhibit macrophage maturation (ie CD16 expression), M2 polarization, and to augment T cell responses will be tested alone and in combination with pharmacologic strategies in the other two projects: cFMS tyrosine kinase inhibitors (Project 2) and NK-1R specifc antagonist (aprepitant) (Project 3). Specific Aim 2: To test the hypothesis that pharmacologic intervention with a lead compound targeting adenosine metabolism (CD39, CD73 or Ador2B antagonists), alone and in combination with tyrosine kinase inhibitors and/or aprepitant, will restore normal monocyte/macrophage dynamics and phenotype, virus specific immune responses in SIV infected rhesus macaques in the presence and absence of cART therapy, and reduce CNS and lymphoid viral reservoirs. In our preliminary studies we demonstrate increased production of CD16+/BrdU positive monocytes in SIV infected animals the periphery at 3 days post BrdU administration. Further, we observe increased T cell clearance in SIV infected animals and importantly a correlation between the rate of T cell turnover and the rate of production of CD16+/BrdU+ positive cells. In our studies performed in this Aim we will select the best drug candidate (lead compound) based on in vitro studies performed in Aim 1, information regarding bioavailability, toxicity, and its potential to synergize with compounds developed in Projects 2 and 3. Here we will investigate the ability of pharmacologic intervention with our lead compound to normalize CD16+/BrdU monocyte and T cell turnover in vivo in SIV infected macaques treated with this compound alone, or in the presence of cART treatment. These studies targeting ATP metabolism to address the persistence of HIV infected reservoirs in the CNS should provide a framework for therapeutic/elimination strategies based on restoration of immune responses, and the interaction of this and other strategies targeting M2 polarization.

PROJECT SUMMARY The Administrative Core is an integral component of this Program Project Grant of this Integrated Pre- Clinical/Clinical Program (IPCP). The Core will have primary responsibility for all financial and scientific oversight management of all projects and cores. The Administrative Core will act as an extension of the Department of Neuroscience Administrative Office to maintain all accounting records. The Administrative Core will facilitate communication between projects and cores, data sharing and publication, subcontract agreements, inter-institutional consortium and intellectual property and technology transfer agreements. In addition to its financial and scientific responsibilities, the core will also have various administrative responsibilities. The administrative coordinator, at Temple University, will work closely with Dr. Rappaport as well as Dr. Douglas' administrative assistant to carry out scheduling the monthly Executive Committee meetings, Internal Advisory Committee Meetings (every six months), Steering Committee (annually). Scientific Advisor Panel (external, annually), and IPCP meeting (annually) including SAP membership as well as NIMH Program Directors to review progress, and make necessary adjustments in consultation and concordance with NIMH Program. There will be close and frequent contact between Dr. Rappaport and Dr. Douglas, as well as the administrative coordinator to this Core and Dr. Steven D. Douglas' assistant, at the Children's Hospital of Philadelphia, a Research Institute. These interactions will serve to facilitate and coordinate the activities of the Program Project's Temple and CHOP components of this IPCP's Projects and Cores. In addition, this Core will interact with the NIMH funded P30s at Temple and CHOP/PENN, whose directors are members of the Internal Advisory Committee. The Administrative Core will be responsible for making all necessary meeting and travel arrangements and hotel accommodations for committee members for core participants of the Executive Committee, Internal Advisory Committee, Steering Committee, Scientific Advisory Panel, and IPCP Committee Meetings. The Core will prepare all financial, scientific, and meeting minutes and reports, as well as maintain records of all correspondence, etc. The Core will prepare the annual non-competing continuation progress reports. The Administrative Core will also maintain records on all institutional protocols regarding the use of human subjects, vertebrate animals, biological materials, technology transfer and patent usage, etc. The Administrative Core will closely collaborate with the Project and Core Leaders on a regular basis to facilitate communication and collaboration, as well as the receipt and exchange of all biological specimens utilized within the individual laboratories.

? DESCRIPTION (provided by applicant): This application is the second competing renewal of a T32 program initially funded in 2005 and initiated in 2006. The proposed Research Training Program in Experimental Medicine and Pathology is designed to prepare veterinarians for independent careers in biomedical research. All trainees (n = 6/year) will be at the postdoctoral level with appointments as Research Fellows in appropriate departments of Tulane University or Louisiana State University. The objective of the program remains to prepare veterinarians for independent careers in biomedical research where they can apply their unique constellation of skills to translational research that will impact human and animal health. This training grant is joint venture between Tulane University Health Sciences (includes the School of Medicine, School of Public Health and Tropical Medicine and the Tulane National Primate Research Center-TNPRC) and Louisiana State University (LSU) Schools of Medicine (LSUSOM) and Veterinary Medicine (LSUSVM) with the TNRPC and LSUSVM having the lead roles. Research projects performed while supported by this training grant can be used towards fulfilling the requirements of a PhD at either Tulane or LSU. Training will include instruction in aspects of comparative medicine, pathology and microbial pathogenesis and broad exposure to state-of-the-art investigative tools needed for molecular-mechanism-based biomedical research using animal models. Funding provided by this application will support mentored research training emphasizing bench and translational research using multidisciplinary methods and critical thinking in experimental design, data interpretation, oral and written communication skills and ethical conduct of research. Trainees also receive instruction in preparation of NIH grant applications and will be expected to submit an NIH grant (K01, K08 or R21). The program consists of diverse, highly skilled and productive research mentors and is guided by experienced leadership including an internal steering committee and an external advisory committee. Measure of success include: i) recruitment and retention of trainees to fill all available positions, ii) attainment of a PhD by all 10 eligible trainees with seven of those individuals now in junior faculty positions in academia or scientist positions in industry or government, and iii) an average of 4 publications/trainee from the training period.

ABSTRACT Alzheimer?s disease (AD) is an extremely important problem with devastating effects on elderly, presently affecting over 5 million persons in the United States. The number of persons with AD is projected to rise dramatically over the next several decades, creating an enormous public health and financial crisis. Thus far, the therapeutic strategies derived from mouse models of AD have universally failed in clinical trials. It has become apparent that the mouse models of AD do not adequately represent the salient features and risk factors associated with AD development and likely then, may not be able to deliver translational products from their studies. Here we propose to develop a model for the study of AD in rhesus macaques. Non human primates such as rhesus macaques and African Green monkeys do develop AD, naturally, with advance aging, unlike mice, however the paucity of such aged animals currently precludes their utility fir AD study. Here we investigate the hypothesis that nicotinamide adenine dinucleotide (NAD+) controls aging, protects against AD, however declines with age. Here we propose to investigate NAD+, and related process and metabolites involved in aging, early development of Alzheimer?s disease, including NAD+ metabolism, insulin resistance, and additional mechanisms involved in AD pathogenesis such as platelet activation and the kynurenine pathway in the context of normal aging in male and female macaques across the life span. We will further determine the ability of the anti-cancer drug, FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT or visfatin) a rate limiting step in NAD+ synthesis, to deplete NAD+ in rhesus macaques. We anticipate that this approach will lead to the acceleration of aging, reveal features of Alzheimer?s and facilitate future studies regarding AD pathogenesis and therapeutics development in this animal model. Our approach represents a fairly thorough interrogation of this hypothesis, involving the investigation of analytes associated with AD development, imaging studies, transcriptomics, and immunohistochemical studies.