Stephanie J. Bissel

Experimental Mycobacterium tuberculosis Infection of Cynomolgus Macaques Closely Resembles the Various Manifestations of Human M. tuberculosis Infection

ABSTRACT Nonhuman primates were used to develop an animal model that closely mimics human Mycobacterium tuberculosis infection. Cynomolgus macaques were infected with low doses of virulent M. tuberculosis via bronchoscopic instillation into the lung. All monkeys were successfully infected, based on tuberculin skin test conversion and peripheral immune responses to M. tuberculosis antigens. Progression of infection in the 17 monkeys studied was variable. Active-chronic infection, observed in 50 to 60% of monkeys, was characterized by clear signs of infection or disease on serial thoracic radiographs and in other tests and was typified by eventual progression to advanced disease. Approximately 40% of monkeys did not progress to disease in the 15 to 20 months of study, although they were clearly infected initially. These monkeys had clinical characteristics of latent tuberculosis in humans. Low-dose infection of cynomolgus macaques appears to represent the full spectrum of human M. tuberculosis infection and will be an excellent model for the study of pathogenesis and immunology of this infection. In addition, this model will provide an opportunity to study the latent M. tuberculosis infection observed in ∼90% of all infected humans.

YKL-40, a Marker of Simian Immunodeficiency Virus Encephalitis, Modulates the Biological Activity of Basic Fibroblast Growth Factor

Human immunodeficiency virus encephalitis causes dementia in acquired immune deficiency syndrome patients. Using proteomic analysis of postmortem cerebrospinal fluid (CSF) and brain tissue from the simian immunodeficiency virus primate model, we demonstrate here a specific increase in YKL-40 that was tightly associated with lentiviral encephalitis. Longitudinal analysis of CSF from simian immunodeficiency virus-infected pigtailed macaques showed an increase in YKL-40 concentration 2 to 8 weeks before death from encephalitis. This increase in YKL-40 correlated with an increase in CSF viral load; it may therefore represent a biomarker for the development of encephalitis. Analysis of banked human CSF from human immunodeficiency virus-infected patients also demonstrated a correlation between YKL-40 concentration and CSF viral load. In vitro studies demonstrated increased YKL-40 expression and secretion by macrophages and microglia but not by neurons or astrocytes. We found that YKL40 displaced extracellular matrix-bound basic fibroblast growth factor (bFGF) as well as inhibited the mitogenic activity of both fibroblast growth factor receptor 1-expressing BaF3 cells and bFGF-induced axonal branching in hippocampal cultures. Taken together, these findings demonstrate that during lentiviral encephalitis, YKL-40 may interfere with the biological activity of bFGF and potentially of other heparin-binding growth factors and chemokines that can affect neuronal function or survival.

A Computationally Optimized Hemagglutinin Virus-Like Particle Vaccine Elicits Broadly Reactive Antibodies that Protect Nonhuman Primates from H5N1 Infection

BACKGROUND Highly pathogenic H5N1 avian influenza viruses continue to spread via waterfowl, causing lethal infections in humans. Vaccines can prevent the morbidity and mortality associated with pandemic influenza isolates. Predicting the specific isolate that may emerge from the 10 different H5N1 clades is a tremendous challenge for vaccine design. METHODS In this study, we generated a synthetic hemagglutinin (HA) on the basis of a new method, computationally optimized broadly reactive antigen (COBRA), which uses worldwide sequencing and surveillance efforts that are specifically focused on sequences from H5N1 clade 2 human isolates. RESULTS Cynomolgus macaques vaccinated with COBRA clade 2 HA H5N1 virus-like particles (VLPs) had hemagglutination-inhibition antibody titers that recognized a broader number of representative isolates from divergent clades as compared to nonhuman primates vaccinated with clade 2.2 HA VLPs. Furthermore, all vaccinated animals were protected from A/Whooper Swan/Mongolia/244/2005 (WS/05) clade 2.2 challenge, with no virus detected in the nasal or tracheal washes. However, COBRA VLP-vaccinated nonhuman primates had reduced lung inflammation and pathologic effects as compared to those that received WS/05 VLP vaccines. CONCLUSIONS The COBRA clade 2 HA H5N1 VLP elicits broad humoral immunity against multiple H5N1 isolates from different clades. In addition, the COBRA VLP vaccine is more effective than a homologous vaccine against a highly pathogenic avian influenza virus challenge.

Astrocyte and Macrophage Regulation of YKL-40 Expression and Cellular Response in Neuroinflammation

Numerous inflammatory conditions are associated with elevated YKL‐40 expression by infiltrating macrophages. Thus, we were surprised to observe minimal macrophage and abundant astrocyte expression of YKL‐40 in neuroinflammatory conditions. The aims of the current study were to better delineate this discrepancy, characterize the factors that regulate YKL‐40 expression in macrophages and astrocytes and study whether YKL‐40 expression correlates with cell morphology and/or activation state. In vitro, macrophages expressed high levels of YKL‐40 that was induced by classical activation and inhibited by alternative activation. Cytokines released from macrophages induced YKL‐40 transcription in astrocytes that was accompanied by morphological changes and altered astrocytic motility. Because coculturing of astrocytes and macrophages did not reverse this in vitro expression pattern, additional components of the in vivo central nervous system (CNS) milieu must be required to suppress macrophage and induce astrocyte expression of YKL‐40.

YKL-40 expression in traumatic brain injury - an initial analysis

YKL-40 (chitinase 3-like protein 1) is expressed in a broad spectrum of inflammatory conditions and cancers. We have previously reported that YKL-40 levels are elevated in the cerebrospinal fluid (CSF) of macaques and humans with lentiviral encephalitis, as well as multiple sclerosis (MS). The current study assessed temporal CSF YKL-40 levels in subjects with severe traumatic brain injury (TBI; Glasgow Coma Scale [GCS] score <or=8). We also evaluated temporal expression of YKL-40 after parasagittal controlled cortical impact (CCI) injury over the parietal cortex (2.8 mm deep, 4 m/sec). We demonstrate that CSF YKL-40 levels are elevated after acute TBI, and that YKL-40 levels are higher in patients who died following injury than in patients who survived. YKL-40 levels significantly correlate with CSF levels of inflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), as well as the inflammatory marker C-reactive protein (CRP). After CCI, in situ hybridization (ISH) showed that YKL-40 transcription is primarily associated with reactive astrocytes in pericontusional cortex. Tissue YKL-40 transcription time course analysis after CCI showed that YKL40 transcription in astrocytes began 1 day after injury, remained elevated for several days, and then declined by day 12. Similarly to our temporal CSF measurements in humans, YKL-40 induction after CCI is coincident with IL-1beta expression. Taken together these findings demonstrate that YKL-40 is induced in astrocytes during acute neuroinflammation, is temporally related to inflammatory mediator expression, and may be a useful biomarker for understanding secondary injury and for patient prognosis.

PET imaging of brain macrophages using the peripheral benzodiazepine receptor in a macaque model of neuroAIDS.

HIV infection in humans and simian immunodeficiency virus (SIV) infection in macaques result in encephalitis in approximately one-quarter of infected individuals and is characterized by infiltration of the brain with infected and activated macrophages. 1-(2-chlorphenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide (PK11195) is a ligand specific for the peripheral benzodiazepine receptor abundant on macrophages and is expressed in low levels in the noninfected brain. We hypothesized that positron-emission tomography (PET) with the carbon-11-labeled, R-enantiomer form of PK11195 ([(11)C](R)-PK11195) could image brain macrophages and hence the development of encephalitis in vivo. [(11)C](R)-PK11195 binding was assessed in the brain using PET in 11 SIV infected macaques, six of which showed increased binding in vivo. Postmortem examination of the brain in these six macaques demonstrated encephalitis, while macaques that did not show an increase in [(11)C](R)-PK11195 binding did not develop SIV encephalitis. Brain tissue from SIV encephalitic macaques also showed increased [(3)H](R)-PK11195 binding compared with binding in nonencephalitic macaques. Increased PK11195 binding in vivo and in postmortem brain tissue correlated with abundance of macrophages but not astrocytes. Our results suggest that PET [(11)C](R)-PK11195 imaging can detect the presence of macrophages in SIV encephalitis in vivo and may be useful to predict the development of HIV encephalitis and in studies of the pathogenesis and treatment of HIV dementia.

Antibody breadth and protective efficacy are increased by vaccination with computationally optimized hemagglutinin but not with polyvalent hemagglutinin-based H5N1 virus-like particle vaccines.

ABSTRACT One of the challenges for developing an H5N1 influenza vaccine is the diversity of antigenically distinct isolates within this subtype. Previously, our group described a novel hemagglutinin (HA) derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This COBRA HA, when used as an immunogen, elicits a broad antibody response against H5N1 isolates from different clades. In this report, the immune responses elicited by the COBRA HA virus-like particle (VLP) vaccine were compared to responses elicited by a mixture of VLPs expressing representative HA molecules from clade 2.1, 2.2, and 2.3 primary H5N1 isolates (polyvalent). The COBRA HA VLP vaccine elicited higher-titer antibodies to a panel of H5N1 HA proteins than did the other VLPs. Both COBRA and polyvalent vaccines protected vaccinated mice and ferrets from experimental infection with highly lethal H5N1 influenza viruses, but COBRA-vaccinated animals had decreased viral replication, less inflammation in the lungs of mice, and reduced virus recovery in ferret nasal washes. Both vaccines had similar cellular responses postchallenge, indicating that higher-titer serum antibodies likely restrict the duration of viral replication. Furthermore, passively transferred immune serum from the COBRA HA VLP-vaccinated mice protected recipient animals more efficiently than immune serum from polyvalent-vaccinated mice. This is the first report comparing these two vaccine strategies. The single COBRA HA antigen elicited a broader antibody response and reduced morbidity and viral titers more effectively than a polyvalent mixture of primary H5N1 HA antigens.

Macrophages Relate Presynaptic and Postsynaptic Damage in Simian Immunodeficiency Virus Encephalitis

Neurodegeneration observed in lentiviral-associated encephalitis has been linked to viral-infected and -activated central nervous system macrophages. We hypothesized that lentivirus, macrophages, or both lentivirus and macrophages within distinct microenvironments mediate synaptic damage. Using the simian immunodeficiency virus (SIV)-infected macaque model, we assessed the relationship between virus, macrophages, and neurological damage in multiple brain regions using laser confocal microscopy. In SIV-infected macaques with SIV encephalitis (SIVE), brain tissue concentrations of SIV RNA were 5 orders of magnitude greater than that observed in nonencephalitic animals. In SIVE, staining for postsynaptic protein microtubule-associated protein-2 was significantly decreased in the caudate, hippocampus, and frontal cortical gray matter compared to nonencephalitic controls, whereas staining for presynaptic protein synaptophysin was decreased in SIV-infected macaques with and without encephalitis. These data suggest that presynaptic damage occurs independent of pathological changes associated with SIVE, whereas postsynaptic damage is more tightly linked to regional presence of both activated and infected macrophages.

Animal models of Rift Valley fever virus infection.

Emerging and naturally occurring infectious diseases from bacterial and viral sources are constantly threatening humans and livestock. Recently, a variety of infectious diseases have emerged into previously disease-free areas, resulting in new epidemics. Consequently, governmental agencies and researchers in the area of biomedical research have started designing ways to prevent their further spread. Ongoing research activities are focused on developing therapeutic and prophylactic interventions against these emerging infections. Development and evaluation of vaccines, diagnostics and treatments often depend on the development of appropriate animal models to determine the efficacy of new therapeutic agents. In addition, animal models are necessary to understand the basic pathobiology of infection. In this minireview, the current animal models used for one of these emerging infectious diseases, Rift Valley fever virus (RVFV), and the specifics of infection and pathology associated with each model are discussed.

Human Immunodeficiency Virus Infection of the Brain: Pitfalls in Evaluating Infected/Affected Cell Populations

Monocyte/macrophages and CD4 T‐cells are the primary hematopoietic targets of productive HIV infection. In the brain, potential cellular targets for HIV infection include perivascular and parenchymal macrophages/microglia, oligodendrocytes, endothelia, neurons, and astrocytes. We examine evidence of productive and non‐productive infection for each cell type in the brains of HIV‐infected patients with and without HIV encephalitis. Despite the voluminous literature and substantial experimental effort over the past two decades, evidence for productive infection of any brain cell other than macrophages is left wanting.