Joseph Mankowski - US grants

DESCRIPTION: This is an amended KO1 application requesting five years of support to provide research training by using an SIV macaque animal model to study the role of the BBB in the development of encephalitis and AIDS dementia. The candidate's preliminary data have demonstrated that neurovirulent but not non-neurovirulent strains of SIV productively infect brain microvessel EC both in vivo and in vitro. This result suggests that the neuroendothelial cells may play an important role in the pathogenesis of SIV encephalitis, and that compromise of CNS EC function may play a pivotal role in BBB disturbances. Furthermore, infection of EC, and alterations in the EC expression of cell adhesion molecules (CAMs) may also contribute to a loss of BBB integrity. The central hypothesis of this application is that BBB dysfunction plays a crucial role in the development of encephalitis and AIDS-related dementia. The first Specific Aim of this application is to assess BBB integrity during acute, latent, and chronic stages of SIV infection to determine whether loss of BBB function is associated with CNS viral load, and CNS pathology. The second Specific Aim is to examine the dynamics of EC infection during disease progression in order to determine if EC infection is associated with altered BBB integrity, increased CNS viral load, and CNS pathology. For both Aims 1 and 2, in vivo analysis employing immunohistochemistry, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) done in situ, will be performed on CNS tissues from animals at various stages of disease. CNS microvessel isolation will permit further characterization of BBB integrity and EC infectivity. The third Specific Aim is to determine if CAMs mediate the interaction between EC and infected and uninfected peripheral blood lymphocytes and monocyte/macrophages during disease progression. Adhesion will be examined both in vivo and in vitro to define the functional role of CAMs during disease progression. Overall, these studies are intended to contribute to the understanding of the role the BBB plays in the development of lentiviral encephalitis.

[unreadable] DESCRIPTION (provided by applicant): HIV-associated cardiomyopathy is a frequent and serious HIV complication, yet the underlying pathophysiology is poorly understood. Intriguingly, prior studies examining SIV as model for neuroAIDS have strongly implicated macrophage infiltration and activation as a key mechanism underlying neurodegeneration. A similar process may occur in the heart and, if so, trigger activation of matrix metalloproteinases that play an important role in chamber remodeling. Accordingly, the central hypothesis of this proposal is that functional cardiac impairment that develops in SIV-infected macaques results from both increased synthesis and activation of selective matrix metalloproteinases (MMPs) associated with macrophage infiltration and activation driven by SIV infection in the heart. The proposed studies will both define the temporal course of decline in cardiac function and its relationship to macrophage activation, SIV replication, and MMP activity, and determine the primary signaling cascades activating the MMPs that play a role in development of cardiomyopathy. There are 3 specific aims: Aim One: To determine the evolution of cardiac dysfunction in SIV-infected macaques based on comprehensive serial echo Doppler and pressure-volume relation analysis and to define the relationship between cardiac dysfunction and inflammatory responses in the heart including myocardial macrophage activation. Host inflammatory responses and cardiomyocyte damage will be measured in endomyocardial biopsy and postmortem tissue samples by immunostaining and quantitative image analysis to compare with cardiac functional status. Aim Two: To determine whether replication of macrophage-tropic SIV strains in the heart is a prerequisite for the development of cardiac dysfunction. To establish the relationship between viral replication and cardiac disease, we will measure myocardial viral load by real-time RT-PCR and identify the predominant replicating viral genotypes in the heart to compare with inflammatory responses including activation of myocardial macrophages, matrix metalloproteinase (MMP) production, and severity of cardiac dysfunction. Aim Three: To determine whether activation of matrix metalloproteinases- specifically gelatinases MMP2 and MMP9, interstitial collagenases MMP1 and MMP13, and MMP12 (macrophage metalloelastase)-are stimulated by SIV-infection of macrophages in the heart and lead to cardiac dysfunction. Tissue inhibitors of MMPs (TIMPs) will also be quantified, and net enzyme protease activity assessed by in situ assay. In vitro studies will test whether a similar profile of MMP synthesis and activation can be produced by SIV-infected cultured macrophages to define the role of specific SIV genotypes present in dysfunctional hearts. These studies will set the stage for performing interventive studies in SIV-infected macaques using approaches to target specific MMPs to prevent cardiac dysfunction. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Peripheral neuropathy is the most common neurological complication associated with HIV-1 infection, affecting over one-third of HIV-infected individuals with AIDS. In addition, studies have found a high prevalence of HIV sensory neuropathy in HIV-infected patients receiving highly active antiretroviral therapy. Although HIV-induced damage to the peripheral nervous system is a frequent and debilitating consequence of infection, its pathogenesis is incompletely understood. To study this disease, we established an SIV/macaque model in which over 90 percent of animals develop PNS changes closely resembling those seen in HIV-infected individuals with distal sensory neuropathy, including inflammation of the dorsal root ganglia with abundant replication of SIV in macrophages and neuronal loss, sural nerve inflammation, and reduction in the number of epidermal nerve fibers in the feet. This constitutes the first primate model of HIV-induced peripheral neuropathy. Our goal is to use this model to dissect the pathogenesis and the underlying molecular basis of HIV-induced PNS disease. Our hypothesis is that HIV first replicates in macrophages within the DRG in the PNS, inducing a cascade of viral and macrophage-produced neurotoxic products, which activate p38 MAPK signaling pathways in somatosensory neurons that trigger sodium channel dysregulation. To address this hypothesis, we have proposed three Aims: Aim 1 is to determine whether SIV-induced PNS disease is initiated by replication of gangliotropic viruses that trigger production of neurotoxic viral and macrophage gene products in the DRG of SIV- infected macaques and to determine whether neurovirulent viruses remain latent in the DRG when no active virus replication is detected in DRG. Aim 2 is to determine the order in which components of the pain pathway, including DRG, sensory fibers in peripheral nerve, and epidermal nerve fibers are damaged in DSP. Aim 3 is to determine whether a) SIV-induced PNS disease is associated with altered conductive properties of unmyelinated C fibers and alterations in expression levels and distribution of sodium channels in DRG neurons, and b) to determine whether active replication of SIV in DRG macrophages induces activation of p38 in DRG neurons thereby modulating the expression and location of sodium channels in DRG neurons. The goal of these comprehensive, integrated studies of HIV sensory neuropathy in our novel SIV primate model is to advance the understanding of HIV-SN pathogenesis to foster new therapeutic approaches.

DESCRIPTION (provided by applicant): HAART has dramatically changed the HIV epidemic, delaying disease, prolonging life and altering the nature of HIV-associated neurocognitive disorders (HAND) from overt dementia to cognitive/motor disorders. Antiretroviral drugs have differing capacities to penetrate into the CNS. It is not clear from HAART therapy in humans whether CNS-penetrant HAART improves CNS outcomes by reducing viral replication and inflammation in the CNS or paradoxically contributes to long term CNS damage due to higher CNS levels of potentially neurotoxic drugs. We developed and characterized a rigorous SIV macaque model of combined antiretroviral therapy (cART) that is very similar to HAART in HIV-infected individuals, combining classes of antiretroviral drugs that have low CNS penetrance. The value of this model is: 1) Both CD4+ T cells and monocyte/macrophages are infected, 2) tissues including brain and spleen harbor virus in HAART-treated macaques, accurately modeling HAART in HIV-infected humans, 3) the number of latently infected resting CD4+ cells in blood and lymphoid tissues is comparable to that in HIV-infected patients on HAART. These advantages demonstrate the model's ability to study latent viral reservoirs in brain and the peripheral nervous system. Virus replication in brain is detectable at low levels using a sensitive single copy assay; SIV DNA levels were comparable to untreated SIV-infected macaques, and there were ongoing inflammatory changes in brain. We now propose to study a SIV model using CNS-penetrant cART (pcART) to determine its impact and compare it directly with non-CNS-penetrant ART (ncART) for ability to 1) control viral replication& inflammation 2) reduce viral DNA reservoirs in brain 3) preserve markers of neuronal function. We will also examine the relative ability of pcART versus ncART to prolong time to virus reactivation after stopping cART. Our hypothesis is that CNS-penetrant cART will be more effective than non-penetrant cART in reducing residual viral replication in CSF and CNS, in reducing viral reservoirs in brain, and potentially other tissues due to increased penetration, as measured by viral DNA and residual virus replication, in reducing CNS inflammation, and in delaying virus reactivation upon withdrawal of cART. However, CNS-penetrant cART might also have neurotoxic effects with increased neuronal damage in both CNS and PNS. We will: 1) Characterize and compare decay kinetics of SIV in plasma and CSF using an optimized CNS-penetrant cART (pcART) to non- penetrant cART (ncART) in our SIV macaque model; quantitate and compare residual virus replication in plasma, PBMCs, CSF and tissues in pcART versus ncART-treated animals; 2) Examine viral latency in CD4+T cells and monocytes in blood and CD4+T cells and macrophages in tissues of SIV-infected macaques treated with pcART & ncART; 3) Measure CNS and PNS markers of neuronal function in SIV-infected macaques with pcART & ncART regimens to evaluate neuroprotection and neurotoxicity; 4) Compare ability of pcART versus ncART to delay or prevent reactivation of virus from reservoirs including the CNS in SIV-infected macaques.

DESCRIPTION (provided by applicant): Combined antiretroviral therapy (cART) has dramatically changed the HIV epidemic, delaying disease and prolonging life. With increasing emphasis on development of strategies to eradicate HIV from latent reservoirs including the CNS, it is critical to evaluate therapeutic approaches in established animal models of HIV latency. To do so, we developed and characterized a SIV model of cART that reduced viral load in peripheral blood and cerebrospinal fluid to undetectable levels. The value of this model is : 1) CD4+ T cells and monocyte/ macrophages are infected, 2) tissues including the brain harbor latent viral DNA, and 3) the number of latently infected resting CD4+ cells in the blood and lymphoid tissues is comparable to that in HIV-infected patients on cART. While SIV RNA in brain was dramatically reduced by cART, SIV DNA levels in brain were unchanged compared to untreated SIV-infected macaques and inflammatory markers remained elevated. In sum, our studies illustrate that cART that suppresses CSF and plasma viral load does not target the CNS latent DNA reservoir. CCR5 inhibitors are promising anti-HIV drug candidates with potential beneficial CNS effects. The CCR5 inhibitor maraviroc (MVC) has high CNS penetrance (CPE = 1.0) and minimal neurotoxicity in comparison with other classes of antiretrovirals. As R5-tropic HIV predominates in the CNS and intermittent HIV replication may persist in the brain despite cART, MVC treatment could block infection of additional cells in the CNS. MVC also may dampen immune activation of resident effector cells in the brain, including microglia and astrocytes, and decrease recruitment of leukocytes to the CNS. Provocative SIV studies by our group evaluated the impact of CCR5 inhibition on SIV-induced CNS damage. In SIV-infected rhesus macaques, maraviroc monotherapy significantly reduced CNS SIV DNA levels and lowered key CNS inflammatory responses in sharp contrast with animals treated with cART. Because of these findings, this proposal focuses on intensification of cART therapy in SIV-infected pigtailed macaques by adding maraviroc with the goal of virus eradication. Our hypothesis is that adding the CNS penetrant CCR5 inhibitor maraviroc to cART in SIV- infected macaques will be much more effective than cART alone in A) reducing viral DNA reservoirs in brain and B) delaying virus reactivation upon withdrawal of cART. These effects are attributable to MVC's ability to both block infection of new cellular targets in the CNS and inhibit pro-inflammatory signaling through CCR5. Aim 1 is to determine whether adding MVC to cART reduces viral DNA and impairs reactivation of latent virus in macrophages/ microglia and astrocytes in the CNS and resting CD4+ cells and monocyte/macrophages in peripheral tissues and blood of SIV-infected macaques. Aim 2 will compare the ability of cART+MVC versus cART alone regimens to prevent or delay reactivation of virus from the CNS reservoir in SIV-infected macaques after cessation of therapy. Aim 3 is to determine whether continuing MVC therapy after stopping cART suppresses reactivation of virus from the CNS reservoir or peripheral reservoirs.

PROJECT SUMMARY Veterinary biomedical researchers greatly enhance animal-based research that serves as the key bridge connecting basic science and human-based studies. Veterinarian scientists play crucial roles by performing biomedical research and by developing, characterizing, and using animal models to study human diseases, enhancing rigor and reproducibility. Building on the outstanding foundation in comparative pathophysiology and medicine provided by DVM/VMD curricula, in-depth, rigorous training in biomedical research is ideal to foster research careers for veterinarians. This proposal seeks renewal of support for our highly productive NIH- funded T32 program led by Joseph L. Mankowski, DVM, PhD, to train veterinarians in biomedical research at the Johns Hopkins University School of Medicine. The primary goal of our program is to launch the careers of veterinarian-scientists. We request funding for six DVM/VMD trainee slots/year to provide 3 years of support/trainee. At Johns Hopkins, veterinarian trainees pursue research-intensive training by completing PhDs or research postdoctoral fellowships if they enter the program holding dual degrees (both DVM/VMD and PhD). We have a deep applicant pool to draw from to fill these positions. Our training program offers an outstanding career development opportunity for veterinarians passionate about building careers in biomedical research and becoming innovators and leaders in their fields. The great majority of trainees pursue either research-intensive or research-related careers after completing training.

? DESCRIPTION (provided by applicant): HIV-induced sensory neuropathy (HIV-SN) is a major global health problem as 35 million people are infected with HIV and a considerable percentage of these will develop neuropathy. HIV-SN poses a major burden on the quality of life of affected patients as many suffer from neuropathic pain that is resistant to antiretroviral therapy as well a other drugs commonly used to treat neuropathic pain. Despite the pressing need for novel therapeutics, the development of new treatment strategies has been hampered by our lack of understanding of the mechanisms underlying HIV-induced neuropathy. Using an SIV/macaque model of HIV-neuropathy that closely recapitulates HIV-induced damage, we propose to investigate the underlying cause of HIV-SN by 1) developing tools that allow the use of corneal confocal microscopy to non-invasively track the development of peripheral neuropathy and 2) measuring levels of SIV-induced inflammatory mediators in plasma and dorsal root ganglia. In this macaque model, we will also record neuronal activity from unmyelinated nociceptive afferents in peripheral nerves to investigate whether such afferents develop signs of peripheral sensitization after infection as sensitization could contribute to spontaneous pain and hyperalgesia seen in patients with HIV-SN. In addition to peripheral nociceptive nerve fibers, nociceptive neurons in DRG may be damaged by inflammatory mediators produced locally in DRG or present in the circulation. Using the patch-clamp electrophysiology technique, we will investigate whether nociceptive DRG neurons develop hyperexcitability during SIV infection and whether inflammatory mediator-sensitive voltage-gated sodium channel isoforms play a role. By correlating the findings obtained with the different techniques, we will obtain a better understanding of the mechanisms underlying HIV-sensory neuropathy to develop new strategies for treatment.

Using the SIVmac251-rhesus macaque model of HIV-1 infection, the Animal Core will provide the long-term ART-suppressed SIV/macaque platform for all projects of this proposal. The Animal Core will 1) coordinate all macaque studies across all research projects, and 2) facilitate acquisition and distribution of valuable animal cells and tissue samples among all research teams in the Program Project. This Core will provide the in vivo validation platform and the blood, CSF, and tissue samples and primary cells necessary to achieve the Program Project?s goals through the following Specific Aims: Aim 1. To coordinate and perform all macaque studies to include animal acquisition and MHC screening, SIV inoculation, ART drug administration, longitudinal sampling of blood (for plasma, serum and PBMCs) and CSF, and biopsy sampling of peripheral lymph nodes. Aim 2. To perform comprehensive necropsies to harvest macaque tissues and fluids at study end points and to perform complete pathology evaluation for all animals. Aim 3. To administer anti-rhesus CD4 antibody to deplete the latent resting CD4+ T cell reservoir in SIV-infected ART treated macaques and monitor for evidence of opportunistic infections.

Abstract Despite fully suppressive ART for long periods of time, HIV rebounds in the majority of individuals when ART is interrupted. Studies in this proposal will define and quantitate the functional SIV latent reservoirs in tissue to understand the contributions of both CD4+T cells and macrophages to the functional viral reservoir and viral rebound after ART interruption. The SIVmac251 ART suppressed model will be used to quantitate the functional viral reservoir in CD4+ T cells and macrophages to asses their contribution to viral rebound. Depletion of CD4 cells and reduction of the latent reservoir will be done to address whether CD4 cells are the major HIV reservoir or if latently infected macrophages significantly contribute to HIV rebound. The projects in this Program will 1) Identify the differential contribution of resting CD4+ T cells and resident macrophages to viral rebound after antiretroviral treatment interruption; 2) Identify the source of viral rebound using SIV proviral genome analysis; and 3) Use computational modeling and analyses of SIV virus rebound to understand HIV rebound.

PROJECT SUMMARY HIV infects macrophages in the spinal cord and brain, often leading to clinical symptoms even with antiretroviral treatment (ART). In spinal cord and brain, macrophages (M?s) may serve as long-term cellular reservoirs of latent HIV. Replication competent HIV can emerge from these latent reservoirs if ART is stopped. SIV replicates in both spinal cord and brain at equivalent levels during acute infection; however, the specific cellular targets of SIV in the CNS have not been classified throughout infection, including during latency and rebound after interrupting ART. In contrast with acute infection, viral dynamics are significantly different in these distinct CNS compartments after stopping ART and tracking SIV rebound. SIV RNA is readily detectable in spinal cord in the first weeks after stopping ART; in contrast, most SIV-infected animals do not have detectable SIV RNA in the brain. The disparity in rebound replication between spinal cord and brain may result from unique cellular immune responses by M? and astrocytes in these distinct CNS compartments. Most SIV- infected macaques receiving ART have no detectable SIV RNA in spinal cord or brain, CSF, or plasma. Proviral DNA and immune responses persist nonetheless in both spinal cord and brain. Intriguingly, the nature of the sustained immune response differs: in the spinal cord, elevated expression of both GFAP and CCL2 point to sustained astrocyte activation; in the brain, elevated CD68 and TNF? levels indicate persistent brain M? activation. The central premise of this proposal is that spinal cord M?s serve as a distinct SIV reservoir in the CNS that reactivates rapidly after stopping ART. Cellular neuroimmune responses in the spinal cord differ from the brain during latency and reactivation after stopping ART. In particular, coordinate regulation of spinal cord M? and astrocyte immune responses intrinsically differ from brain responses. Specific Aim 1 is to determine whether spinal cord M?s constitute a unique SIV reservoir in the CNS by identifying SIV-infected CNS cell populations during 1) acute SIV infection, 2) prolonged latency with ART, and 3) viral rebound after stopping ART. Specific Aim 2 will identify the neuroinflammatory responses in the spinal cord and brain during 1) acute SIV infection, 2) prolonged latency on ART, and 3) viral rebound after stopping ART. Gene expression profiling will be performed using nCounter immunoregulatory assays; single cell RNA-seq gene expression profiling performed on single cells isolated from spinal cord and brain including M?s and astrocytes will complement targeted gene profiling. Specific Aim 3 is to determine whether depleting spinal cord and brain M?s during latency and continuing after stopping ART by treatment with the CSF1R inhibitor PLX3397 alters CNS SIV replication during SIV rebound from latency. M? depletion will complement SA1 studies on CNS M?s as latent reservoirs and SA2 studies on spinal cord gene expression profiles in latency and during SIV rebound to advance our understanding of the role that spinal cord M?s play in HIV latency.

Veterinarian scientists greatly strengthen the translational bridge between basic science and clinical medicine. Their dual training endows them with unique expertise that enhances the rigor and reproducibility of research with animal models and improves animal welfare. To succeed in a research career, veterinarians must build on the foundation in comparative medicine gained through their veterinary education by engaging in PhD programs or postdoctoral fellowships that provide rigorous training in biomedical research. This is a path that ideally begins while still a veterinary student, with the student participating in mentored research projects and networking with veterinarian-researcher role models. This proposal seeks to expand existing summer training program opportunities for veterinary students in biomedical research at the Johns Hopkins University (JHU) School of Medicine as a means to recruit more veterinarians to research careers. The proposed T35 Scholars Program will ignite a passion for biomedical research in participating students thorough immersion in a mentored summer research project at our top tier academic medical center. The Department of Molecular and Comparative Pathobiology (DMCP), a basic sciences department in the JHU School of Medicine that serves as the academic home for DVM faculty at JHU, will provide a supportive home base replete with veterinarian- scientist role models from postdoctoral fellows in our T32-funded training program to tenured professors with R01-funded independent research labs. DMCP will additionally host a program of career development activities educating the veterinary students about the careers available to veterinarians in research. We request funding for the proposed 12 week program for 4 summer veterinary student trainee slots / year for the first two years, to expand to 6 slots / year for the final three years. The proposed T35 Scholars Program will complement and integrate into our existing summer programs that have >90% success in preparing veterinary students for research-intensive careers. Through the establishment of a T35 Scholar Program at JHU DMCP, we hope to offer an outstanding opportunity for veterinary students to learn about and prepare themselves to be future veterinarian-scientists as well as leaders in biomedical research.