Serena Spudich - US grants

[unreadable] DESCRIPTION (provided by applicant): The candidate's long-term goal is to understand, then prevent or treat, mechanisms underlying the damaging effects of HIV in the central nervous system (CNS). The proposed research focuses upon events in the CNS during primary HIV infection, defined as the first six months after initial acquisition of virus. HIV enters the CNS in the earliest stages of infection, and during chronic infection the CNS is a site of persistent viral infection. The underlying hypothesis of the proposal is that early neuroinvasion by HIV involves viral trafficking in the setting of host immune activation and allows the establishment of persistent and compartmentalized CNS infection. The candidate will use serial cross-sectional evaluations of individuals during primary infection and during initiation and interruption of therapy to longitudinally define CNS host responses and features of cerebrospinal fluid (CSF) HIV populations. The first aim is to demonstrate the natural history of host CNS responses of immune activation, inflammation and tissue injury through measurement of cerebral metabolites by high-field (4 Tesla) magnetic resonance spectroscopy, CSF cellular and inflammatory changes, and neurobehavioral indices. The second aim is to compare viral loads and molecular characteristics of viral populations between the CSF and plasma, including genotypic and phenotypic resistance, chemokine receptor utilization, and clonal analysis, to demonstrate early establishment of compartmentalized infection. The final aim is to use the above techniques to define the effects of treatment with antiretroviral therapy on the course of primary HIV in the CNS, through cross-sectional, serial studies of individuals initiating treatment for reasons independent of this proposal. Identification of primary HIV infection as a crucial period for establishment of CNS infection and injury and revealing the early effects of treatment in the CNS will profoundly influence treatment strategies in early HIV. The candidate's career development plan will allow her to translate observations from clinical research into improved understanding and treatment of CNS complications of HIV. Participation in a formal Advanced Training in Clinical Research program will foster sophisticated and responsible investigation. Clinical service in neurology will include care of patients with AIDS as well as those with other infectious conditions. Finally, the guidance of Drs. Price, Meyerhoff, and Hecht and support from UCSF's Neurology Department will allow the applicant to excel as an integral member of the academic community. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The proposed research focuses upon events in the central nervous system (CNS) during the earliest weeks and months after initial acquisition of HIV infection, collectively defined as primary HIV infection (PHI). HIV enters the CNS in the earliest stages of infection, and the CNS is a site of persistent viral infection throughout the chronic stages of disease. The underlying hypothesis of this proposal is that initial viral neuroinvasion is important in the neuropathogenesis of HIV, leading to the foundation of persistent and compartmentalized CNS infection, and initiating the process of brain injury. The investigators will longitudinally study 75 subjects presenting during PHI to study the course of host immune and neurological responses and features of cerebrospinal fluid (CSF) HIV quasispecies beginning during this period. The first aim is to understand the relationship between viral burden, early host inflammation, and tissue injury in the CNS, through measurement of CSF markers of inflammation, immune response, and neuronal injury, cerebral metabolites by high-field (4 Tesla) magnetic resonance spectroscopy, and neuropsychological testing. The second aim is to investigate the establishment of compartmentalized CNS infection through use of the heteroduplex tracking assay to detect HIV quasispecies sequence differences between and within CSF and plasma, and through measurement of viral replicative capacity and coreceptor utilization to define the character of early HIV species in each compartment. The final aim is to describe the effect of antiretroviral therapy initiated during PHI on the course of CNS inflammation and neurological responses. This study establishes a cohort for extended follow-up and a repository of banked longitudinal samples for future studies. Our proposed approach will provide crucial information about the clinical importance of early HIV in the nervous system;if immunoactivation-mediated brain injury or the establishment of compartmentalized CNS infection occurs during PHI, early treatment with immune- modulating or antiretroviral medications may provide previously unrecognized long-term neuroprotection. Similarly, detection of beneficial effects of treatment on the CNS during PHI has the potential to profoundly influence treatment strategies in early HIV. The overall goal of this proposal is to ameliorate or prevent HIV-related CNS damage through improved understanding of the early effects and treatment of HIV in the nervous system. Central nervous system (CNS) impairment remains a major complication of HIV-1 infection, and has the potential to affect at least 20% of the 40 million people worldwide who are living with HIV-1. Clarification of the time course of establishment of CNS infection and neurological injury will contribute to an understanding of the significance of the earliest stages of HIV-1 infection in the neuropathogenesis of AIDS. In addition, revealing the early effects of antiretroviral treatment in the CNS may provide a new rationale for initiating antiretroviral therapy in early HIV-1 infection.

DESCRIPTION (provided by applicant): The systemic immune response and central nervous system (CNS) events occurring during acute HIV likely set the stage for chronic HIV-related CNS injury and the establishment of CNS-relevant HIV reservoirs. Just as the earliest systemic features such as peak plasma HIV RNA, level of T-cell activation, and early loss of CD4 cells are crucial determinants of HIV disease trajectory, CNS processes initiated during the earliest stages may critically inform the establishment of a CNS-relevant viral reservoirs, CNS compartmentalized virus, the hosts' ability to control CNS virus, and the long-term CNS consequences of infection. Logistical challenges have lead to heavy reliance on animal data to define the likely CNS events during acute HIV. In this application, we extend existing partnerships with US Army studies underway in Thailand to define these earliest events in humans and determine factors that influence long-term CNS outcomes. This application proposes to provide intensive CNS characterization for 60 Thai subjects enrolled during acute HIV (< 1 month after exposure). In our schema, one-half of subjects will begin HAART immediately after initial assessments for a fixed 18-month course. We will longitudinally characterize CNS clinical events, neurological, neuropsychological, and psychiatric factors, multimodal magnetic resonance imaging, CSF immunology and compartment specific full-genome HIV sequencing to determine how the events that occur in acute HIV impact chronic HIV CNS disorders. We will also determine CNS founder and established viruses and determine if early HAART intervention impacts these relationships. The parent studies include extensive systemic immunological and virological characterization allowing us to determine if the earliest systemic events impact long-term CNS outcomes.

DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) infected patients are now living longer due to potent antiretroviral therapy (ART). Despite the effectiveness of ART for systemic viral suppression, up to half of chronically HIV infected (HIV+) individuals will develop HIV associated neurocognitive disorders (HAND). Increasing efforts have focused on understanding the early pathophysiologic changes within the central nervous system (CNS) initiated during primary HIV infection (PHI, defined as the first year after HIV transmission). It remains unknown if early initiation of ART during PHI could reduce the future incidence of developing HAND. It is imperative that early biomarkers (including cerebrospinal fluid (CSF) and neuroimaging) are developed for understanding the mechanisms of crucial early events that may influence the trajectory of damage in the CNS. Previous studies of PHI have primarily been cross sectional and have focused on a limited number of PHI participants assessed by single modality. We propose a longitudinal study to assess the effects of PHI and early ART on brain structure which integrates structural neuroimaging analyses with blood and CSF measures of infection, inflammation and neurodenegeration, as well as clinical data including exposure to stimulant drugs of abuse to investigate possible mechanisms of brain injury beginning during PHI. Specifically, this study will employ structural neuroimaging methods (diffusion tensor imaging (DTI) and brain volumetrics) to study the CNS mechanisms that underlie the natural history of PHI in the CNS as well as the effects of starting ART in the early stages of infection. Based on our preliminary cross sectional analyses, we hypothesize that structural neuroimaging (DTI and brain volumetrics) will reveal progressive injury in the brain during early infection prior to ART which may be halted in subjects that initiate ART soon after seroconversion. Subjects using methamphetamine may have increased vulnerability to HIV-associated structural injury, tied to inflammation in the brain. This proposal will: (Aim 1) investigate the mechanisms of CNS injury in PHI prior to ART by temporally associating the emergence of abnormalities in structural imaging with laboratory biomarkers and neuropsychological performance. Additionally (Aim 2) we will explore the mechanisms of early ART effects in the CNS through assessment of white matter integrity and brain volumetrics over time in subjects both before and after starting ART. Our long term goal is to provide evidence for the appropriate timing of ART initiation and to evaluate the rationale of possible tailored adjunctive neuroprotective therapies to prevent HAND.

DESCRIPTION (provided by applicant): Central nervous system (CNS) infection of HIV is established early in the course of infection and intervention during the phase of acute HIV infection (prior to antibody seroconversion) provides the best opportunity to prevent the establishment of HIV reservoirs in the CNS. Here we capitalize on an established infrastructure for intensive CNS studies in a unique cohort of acute HIV infection subjects in Bangkok enrolled as part of the ongoing US Military-funded RV254 study. We propose two distinct randomized intervention strategies given in addition to antiretroviral therapy (ART) instituted during the earliest stages of acute HIV infection. The first study will employ immediate adjunctive telmisartan therapy (an antifibrotic/anti-inflammatory angiotensin II receptor blocker) to reduce HIV-associated inflammation and reservoir establishment in the CNS. We hypothesize that telmisartan therapy with ART versus ART alone during acute infection will reduce systemic immune activation and trafficking of activated and HIV-infected cells to the CNS, limiting establishment and persistence of the CNS reservoir of HIV. The second study will assess the CNS effect of delayed adjunctive romidepsin (a histone deacetylase inhibitor) therapy given to activate and kill systemic latently HIV-infected cells. We hypothesize that while romidepsin has a postulated effect of activating latent HIV and purging systemic reservoirs, due to low CNS penetration of romidepsin, romidepsin with ART versus placebo with ART will have limited effect on the CNS reservoir. Finally, we will assess early CNS compartmentalization of HIV species as well as the source of rebound HIV detected in the CNS after interruption of ART in the romidepsin study using ultra-deep sequencing to compare blood and cerebrospinal fluid (CSF) variants prior to ART and after ART interruption. Twenty-one subjects in each study will be randomized 2:1 to intervention versus no intervention and followed for 1.5 years. Careful neuropsychological testing will be performed, and blood, CSF samples and magnetic resonance imaging and spectroscopy will be collected to interrogate brain function and inflammation. These data will significantly advance our understanding of HIV persistence and inflammation in the CNS. The knowledge gained will be critically relevant to the 40 million people worldwide living with HIV, informing novel strategies aimed at viral eradication of HIV and prevention of inflammation in the CNS.

? DESCRIPTION (provided by applicant): Despite extensive clinical, laboratory, and imaging evidence that the central nervous system (CNS) is persistently abnormal in some HIV-infected individuals with systemic viral suppression on combination antiretroviral therapy (cART), the underlying mechanisms of this perturbation are poorly understood, including etiologies, cells and tissues involved, and relation to potential persistence of HIV. In an era of increasing global access to cART, defining perturbations of the CNS on sustained cART and identifying potential interventions is the most salient neurologic issue for the 35 million people currently living worldwide with HIV. The current pilot project exploits recognition of the role of exosomal micro-RNAs (exo-miRNA) in disease pathogenesis to further our understanding of the processes underlying HIV related CNS abnormality detected on long-term suppressive cART. Exosomes are membrane nanovesicles released via exocytosis of donor cells that contain small non-coding miRNAs (exo-miRNAs) that regulate gene expression in target cells. Exo-miRNAs play important roles in disease pathogenesis including regulating immune responses in target tissues. Furthermore, viral exo- miRNAs may reveal HIV persistence, and other specific exo-miRNAs indicate cellular origins. To examine the associations between exo-miRNA in the CNS and cART treated HIV, in Aim 1 we will cross-sectionally profile exo-miRNA in cerebrospinal fluid (CSF) and blood by deep sequencing in individuals on sustained cART and HIV-uninfected comparison study participants. We hypothesize that CSF exo-miRNA associated with inflammation will be more abundant in cART-suppressed participants and that CSF exo-miRNAs will be enriched for inflammatory-mediating miRNAs compared to blood. We will explore whether HIV-derived exo-miRNAs can be detected in CSF, suggesting HIV persistence, and the impact of early versus later initiation of cART on exo-miRNA profiles. In Aim 2 we will investigate the mechanistic relationships between exo-miRNA profiles, immune activation and exhaustion in the CNS and blood, and neurocognitive performance. We hypothesize that in CSF and blood, exo-miRNAs linked to inflammation will correlate with activation of monocytes and T lymphocytes as measured by flow cytometry and soluble markers of immune activation, and that poorer neuropsychological testing performance will associate with inflammatory profiles of miRNA in CSF. Our long-term goal is to provide improved understanding of the causes of CNS perturbation in treated HIV in order to develop targeted adjunctive therapies to ameliorate HIV associated neurocognitive disorder.

The most pressing neurologic priorities relevant to the 37 million people living with HIV (PLWH) worldwide are to identify causes of central nervous system (CNS) dysfunction during virally suppressive combination antiretroviral therapy (cART) and interventions to correct them. Gaps in our understanding of the biological basis of HIV associated neurocognitive disorder (HAND) during cART impede progress in ameliorating neurocognitive impairment in long-term surviving PLWH. More than 20 years ago, reduced synaptic density was recognized as the primary pathology in autopsy specimens from HIV infected donors with mild forms of cognitive impairment at the time of death. Reduction in density of synaptophysin-immunoreactive terminals was identified in early stage impairment, in the absence of classical findings of HIV encephalitis. These compelling findings set the groundwork for subsequent important preclinical in vitro and animal studies revealing further understanding of reduced synaptic density in HIV, including regional vulnerability, contributory mechanisms, and potential interventions. However, confirmation of these findings and further investigation has not been possible to date in living, virologically suppressed humans due to lack of access to brain tissue samples. Additionally, unlike frank neuronal loss, synaptodendritic injury may be reversible. Thus, the ability to detect decreases in synaptic density in living humans and to identify potential mechanisms that correlate to its presence would guide therapeutic approaches and provide a critical biomarker for monitoring effects of novel therapeutics to reduce brain dysfunction in HIV. This application capitalizes on recent unprecedented expansion of imaging technologies to apply the understanding gained by pre-clinical studies to investigation of synaptic density in living humans. We have recently developed a novel radiotracer, 11C?UCB?J, for imaging synaptic density in the human brain using positron- emission tomography (PET). In the proposed research studies, we will apply this breakthrough methodology to explore whether observations of decreased synaptic density in postmortem human samples and animal models will be found living PLWH with suppressed HIV replication. Further exploratory studies will investigate associations between synaptic density and laboratory and clinical measures implicated by preclinical studies, including levels of systemic and CNS immune activation and history of opioid use. Our pilot study validating this modality as a means to detect aberrant synaptic density in cART-treated HIV will have a major impact, setting the stage for future studies of the relationship of synaptic density to clinical outcomes of HAND, and providing a therapeutic target and a biomarker for treatment studies aimed to improve HIV-related injury in the CNS.

Central nervous system (CNS) inflammation is a hallmark of HIV neuropathogenesis in untreated advanced AIDS. During systemically suppressive combination antiretroviral therapy (cART), abnormal inflammation persists, and associates with mild neurological impairment and, in dramatic form, with progressive neurologic disease in symptomatic cerebrospinal fluid (CSF) HIV escape and CD8+ T cell encephalitis. Defining neuroinflammation in exquisite detail, including rare and/or novel populations that distinguish HIV infection during cART, has the potential to provide critical targets for therapeutic intervention for residual neurologic impairment during HIV treatment. The proposed studies capitalize on recent dramatic expansion of technologies and understanding of microfluidics, molecular barcoding, and sequencing to facilitate the precise, unbiased, and high throughput sequencing of RNA expressed in single cells. Thousands of individuals cells in a given tissue type can now be profiled in detail to understand the cellular composition of healthy tissues, and to begin to unravel the cellular disruptions present in disease. We have recently successfully applied massively-parallel single-cell RNA sequencing to transcriptionally profile thousands of cells derived from the CSF at the single cell level. Our preliminary studies demonstrate application of Seq-Well, a portable, low-cost platform for single cell RNA- sequencing designed to be compatible with low-input clinical samples, to analysis of CSF and blood from individuals with well-treated HIV and HIV-uninfected controls. In the current R21 proposal, we seek to apply this breakthrough methodology followed by advanced bioinformatics analysis to demonstrate the utility of single cell RNA sequencing of CSF to study pathogenesis and mechanisms of HIV related injury in the CNS in individuals with HIV on CART. Large datasets generated through high throughput sequencing require meticulous and expert analyses. Our planned thorough bioinformatics approach will provide unprecedented depth of insight into complex processes, and will demonstrate the potential of single cell RNA sequencing of CSF to help to unravel the complexities of neuroinflammation relevant to CNS HIV and numerous other neurological disorders.

We propose the designation of a Yale Neuroscience Clinical Trials Center (Yale INFINITY) as a Clinical Site in the NINDS Network of Excellence in Neuroscience Clinical Trials (NeuroNEXT). INFINITY has been designed to serve as a vibrant and top-enrolling contributor to NeuroNEXT. Yale is the 7th largest hospital in the US, and Yale Neurology is 6th highest in NIH funding. Yale is widely recognized as a leading tertiary care center in neurology with a robust faculty and excellence in clinical research. Principal Investigator Dr. Kevin N. Sheth and Co-Principal Investigator Dr. Serena Spudich bring a complementary set of skills and experience in creating innovative clinical research programs in neurological disease. They have worked together at Yale for years, along with Dr. David Hafler, Neurology chair, and an enthusiastic cadre of faculty across disciplines, to create a highly collaborative environment focused on clinical research, multicenter trials, membership on Institutional Review Boards, clinical trial committees, and extensive mentoring in patient-oriented research. There is robust neurology representation in the Yale NIH-funded CTSA (Yale Center for Clinical Investigation ? YCCI), and faculty have assumed positions of international leadership in their fields. Yale faculty have served as principal investigators in several multicenter studies and trials, including one of the NeuroNEXT trials, the NN103 BEAT MG Study, led by Dr. Richard Nowak, Trial Operations Co-Chair for Yale INFINITY. The Center already has existing infrastructure with an established track record of outreach, high quality patient enrollment, operational efficiency, research training, and communications. The Yale Clinical Site is strengthened by the following characteristics: 1) A pool of talented investigators with nationally recognized clinical research expertise across the entire spectrum of adult and pediatric neurological disorders, 2) a geographically diverse and large pool of patients in Connecticut and Southern New England currently not captured by the existing NeuroNEXT network, many of whom are seen at Yale specifically in order to participate in cutting edge trials, 3) talented teams of investigators with deep experience in clinical trials, recruitment and retention, and disease specific networks, 4) specific and nationally recognized expertise in biomarker development and validation, 5) a large cohort of ongoing NINDS-funded clinical studies to which the Yale Center can contribute to during any relative inactive period, 6) patient and community engagement embedded within the INFINITY leadership, 7) strong commitment from Yale institutional officials for participation in a central IRB and Master Trial Agreements, and 8) institutional commitments in the form of dedicated space and resources, as well as funding for a master's degree in clinical research for NeuroNEXT fellows. The Yale Neurological community is poised to make significant contributions to enhance and further the mission of NINDS and NeuroNEXT to reduce the burden of neurological disease.

This is a proposal to support the Yale Clinical and Epidemiology Research in Neurology (CERN) Training Program. The thematic concept and single focus of the proposed program is to train clinical neurologists in clinical and epidemiological research methods. Yale has an exceptionally strong existing research base in clinical neuroscience investigation. The Department of Neurology is ranked the 9th highest clinical neurology department in NIH funding. The critical mass of mentors with specific expertise in epidemiology and clinical research relevant to the neurosciences has led to a natural expansion of the training opportunities at Yale, fostering the development of the proposed program. The engagement of these mentors will provide an outstanding foundation for this novel research training. Our proposed program encompasses four key neurologic disease areas: cerebrovascular disease, neurodegeneration and aging, neuroinflammation, and epilepsy. The program is supported by 20 carefully-selected faculty across the Schools of Medicine and Public Health (YSPH). Our objective is to provide a closely mentored rigorous research training program for postdoctoral physician scientists to gain education and experience with the tools necessary for investigative academic careers focused on human subjects research. Two new CERN Training Program fellows per year will be selected for a two-year training program from clinically trained candidates with MD degrees and prior neurological specialty training (i.e., neurology residency). Central to the training experience will be pursuit of a project involving clinical or epidemiological research in neurology. In addition to pursuing mentored research projects, trainees will complete core coursework in these areas offered through the YSPH, and will participate in a series of regularly scheduled didactic series, seminars and workshops, and a course on the responsible conduct of research, which are available through the Yale Center for Neuroepidemiology and Clinical Neurological Research (CNE2) and the Yale Center for Clinical Investigation (YCCI). Through a carefully selected didactic and educational program, and close monitoring of trainee progress with a formalized mentoring process, trainees will gain the skills to design and execute rigorous investigative projects, analyze their data, and publish their results. Trainees will be guided both by mentors and YCCI- and YSPH-supported grant-writing workshops, with the goal of having successfully applied for independent career development awards by the conclusion of the fellowship. With abundant resources in the form of study participants, existing cohorts and datasets, outstanding collaborators, and educational opportunities, Yale provides an exemplary environment for clinical and epidemiologic neurologic research. This will be leveraged through this program to train a cadre of leading patient-oriented investigators, with a shared goal of prevention of or reduction of morbidity and mortality associated with neurological disease.

Abstract Opioid use disorder (OUD) and HIV infection are syndemic conditions that independently and synergistically lead to central nervous system (CNS) dysfunction in tens of millions of people globally. However, the cellular circuits altered by OUD and HIV, and their combination, remain elusive. Further, the identities of cell types within the brain that can harbor HIV infection remain controversial. To address this key vexing question of HIV location within the brain and the effects of HIV and OUD on the brain, comprehensive tissue characterization at the single-cell level is needed to identify novel rare cell types, enriched or depleted cellular populations, and cellular circuits tied to pathogenesis. We propose to employ state-of-the-art methodologies in a center at Yale devoted to generating data on Single Cell Opioid Responses in the Context of HIV Discovery (SCORCH), Y-SCORCH. The center assembles a team of investigators at Yale with leading expertise in neurogenomics, HIV biology, neuroscience of addiction, single-cell analytics and consortium science, and a record of existing collaborations. Our TISSUE Component includes plans to sample 20 brains from four donor groups: controls, HIV (HIV+), HIV with OUD (HIV+OUD+), and OUD without HIV (OUD+). For each brain we will study 4 regions (prefrontal cortex, ventral striatum, insular cortex, and amygdala), representing disease-relevant areas for OUD and HIV. Our ASSAY Component will carry out single nucleus RNA sequencing (snRNA-seq) for 5,000-20,000 cells/sample, single-nucleus ATAC-seq (scATAC-seq), and spatial transcriptomics to generate transcriptomic, epigenetic, and spatial atlases for each donor type and each region. In parallel, we will detect HIV transcripts in the HIV+ groups. Our DATA Deposition & Analysis Component will assemble data standards, facilitate dissemination, and integrate our data with existing brain atlases. It will develop pipelines for high-throughput data analysis, including for single nucleus transciptomes, detection of HIV transcripts and for scATAC-seq data, and develop innovative analysis methods. Our Prioritization & Functional VALIDATION Component proposes a process of identification of brain regions and donor types that best differentiate disease states, and describes experiments to validate the findings generated by transcriptomic and epigenetic analyses. Finally, our Research MANAGEMENT Component provides a framework for data sharing with the SCORCH Data Center and broader Consortium and ensures timely progress in achieving our milestones which include generating `omics data from 640 assays. Our Specific Aims are to: (1) Establish a workflow from tissue samples to single-cell data deposited in the data center, (2) Run the combined experimental and computational workflows on procured specimens, and (3) Follow up on large-scale data production with validation and further analysis. Y-SCORCH has established expertise in all approaches necessary to successfully create single-nucleus transcriptomic data to provide a scaffold for future discovery to inform pathophysiological understanding of CNS effects of OUD and HIV.

Project Abstract Despite prolonged suppression of HIV on antiretroviral therapy (ART), eradication or sustained remission of the infection has not been achieved. Low levels of HIV DNA are still detectable in peripheral blood mononuclear cells (PBMC) from people living with HIV (PWH) taking ART, and cells containing rebound-competent virus can reside in sanctuary tissue sites, including lymph nodes, gut, genital tract and the central nervous system (CNS). In a study conducted within the AIDS Clinical Trials Group, we have recently used highly sensitive virologic assays in living donors to detect HIV DNA in cerebrospinal fluid (CSF) cells in up to 50% on long-term ART. Importantly, those with detectable CSF HIV DNA had poorer global cognitive function that those in whom CSF HIV DNA was not detected. We have subsequently shown higher concentrations of HIV DNA in CD4+ T cells from CSF compared to contemporaneous PBMC, and that atypical cell lineages including myeloid cells may be infected in CSF. Finally, we have successfully used single cell transcriptomics to identify unique and rare cell types in the CSF in PWH associated with HIV disease status and have further demonstrated the ability to identify cellular transcripts enriched in PWH versus healthy controls. Critical gaps in understanding CNS HIV persistence include what the characteristics and function of infected immune cells are in CSF, whether HIV proviruses in CSF are intact and genetically compartmentalized compared to proviruses in blood, and how these features relate to neuropsychiatric function of long-term HIV. Since the number of cells present in CSF is low in PWH suppressed on ART, thorough, simultaneous characterization of the immunologic and virologic landscape of the CNS has not been achieved. Using optimized lumbar puncture procedures and novel molecular techniques, we have overcome these obstacles to both rigorously examine the phenotype of CSF cells and thoroughly characterize the size, stability, intactness, and sequence diversity of persistent HIV in CSF compared to blood. We will use single cell technology to measure the transcriptional and cytokine profile of CNS cells combined with novel quantification of intact HIV DNA and single genome sequencing to discover new correlations within the CNS reservoir. Most importantly, we propose to rigorously examine the cognitive function and mental health of people living with HIV using sophisticated implementation of the new NIMH Research Domain Criteria (RDoC) framework, and how differences in neuropsychiatric outcomes relate to specific immunological and virological characteristics of the CNS in a diverse cohort of participants with a range of neuropsychiatric comorbidity.

Summary/Abstract Widespread use of effective antiretroviral therapy (ART) in the past two decades has improved the clinical manifestations and altered the pathology of neurologic disease in people living with HIV (PLWH). However, in the context of this treatment that has successfully prolonged millions of lives, new pressing questions have emerged regarding the etiology of persistent neurological and cognitive impairment that frequently manifests in PLWH on ART. The human brain is a complex and inaccessible organ that, to date, defies comprehensive understanding in the context of homeostasis and disease. Thus, the hallmark neuropathologic finding of HIV associated neurocognitive disorder (HAND), synaptodendritic injury, has thus far only been possible to evaluate in post-mortem human autopsy brains and animal models of HIV. Our group at Yale recently developed and validated a novel positron-emission tomography (PET) radiotracer, [11C]UCB-J, a ligand for the presynaptic vesicular membrane protein synaptic vesicular glycoprotein 2A (SV2A), to image synaptic density in the human brain. The premise of this application is based on our ongoing pilot study which demonstrates that brain SV2A PET successfully identifies regions of reduced synaptic density -- including in a hippocampal- frontostriatal neural circuit that correlates with CNS dysfunction -- in PLWH on ART. In the current proposal, we seek to measure synaptic density longitudinally over 24 months in a larger group of PLWH on ART relative to a matched prospectively enrolled HIV-negative group. We will combine this breakthrough imaging methodology of synaptic density with PET imaging of neuroimmune status (using radioligand [11C]PBR28 targeting the 18-kDa translocator protein, or TSPO, sensitive to microglia) to explore the premise that neuroimmune dysregulation during ART mediates reduced synaptic density. Finally, we aim to longitudinally integrate these measures with neurocognitive and laboratory assessments in parallel processing models that allow us to dissect mechanisms and clinical outcomes of HAND. Our application is based on robust and compelling preliminary data that demonstrates the utility of SV2A PET imaging in identifying regions of reduced synaptic density in PLWH on ART. Our proposed project will generate powerful clinical-translational support for potentially reversible brain alterations in synaptic density, and their relation to microglia levels and clinical and biological measures in PLWH. This program will set the stage for identification of therapeutic targets and provide a biomarker for interventional studies aimed to improve HIV-related injury in the CNS.