Persistence and pathogenesis of the neurotropic polyomavirus JC

Many neurological diseases of the central nervous system (CNS) are underpinned by malfunctions of the immune system, including disorders involving opportunistic infections. Progressive multifocal leukoencephalopathy (PML) is a lethal CNS demyelinating disease caused by the human neurotropic polyomavirus JC (JCV) and is found almost exclusively in individuals with immune disruption, including patients with human immunodeficiency virus/acquired immunodeficiency syndrome, patients receiving therapeutic immunomodulatory monoclonal antibodies to treat conditions such as multiple sclerosis, and transplant recipients. Thus, the public health significance of this disease is high, because of the number of individuals constituting the at‐risk population. The incidence of PML is very low, whereas seroprevalence for the virus is high, suggesting infection by the virus is very common, and so it is thought that the virus is restrained but it persists in an asymptomatic state that can only occasionally be disrupted to lead to viral reactivation and PML. When JCV actively replicates in oligodendrocytes and astrocytes of the CNS, it produces cytolysis, leading to formation of demyelinated lesions with devastating consequences. Defining the molecular nature of persistence and events leading to reactivation of the virus to cause PML has proved to be elusive. In this review, we examine the current state of knowledge of the JCV life cycle and mechanisms of pathogenesis. We will discuss the normal course of the JCV life cycle including transmission, primary infection, viremia, and establishment of asymptomatic persistence as well as pathogenic events including migration of the virus to the brain, reactivation from persistence, viral infection, and replication in the glial cells of the CNS and escape from immunosurveillance. Ann Neurol 2015;77:560–570     

Overview of the cellular immunity against JC virus in progressive multifocal leukoencephalopathy

The human polyomavirus JC (JCV) infects most healthy adults without causing any disease. In the setting of severe deficit of cell-mediated immunity, such as in acquired immunodeficiency syndrome (AIDS), malignancies or in organ transplant recipients, JCV can reactivate and cause progressive multifocal leukoencephalopathy (PML), a deadly demyelinating disease of the central nervous system. The humoral immune response, measured by the presence of virus-specific immunoglobulin G (IgG) in the blood or by intrathecal synthesis of IgG in the cerebrospinal fluid (CSF), is unable to contain the progression of PML. CD4+ T lymphocytes recognize extracellular viral proteins that have been degraded into peptides through the exogenous pathway and presented on major histocompatibility complex (MHC) class II molecules at the surface of antigen-presenting cells. Consistent with their underlying immunosuppression, the proliferative response of CD4+ T lymphocytes to mitogens or JCV antigens is reduced in PML patients. CD8+ cytotoxic T lymphocytes recognize intracellularly synthesized viral proteins that have been degraded into peptides through the endogenous pathway, and presented on MHC class I molecules at the surface of virus-infected cells. One of such JCV peptide, the VP1(p100) ILMWEAVTL, has been characterized as a cytotoxic T lymphocyte (CTL) epitope in HLA-A *0201 + PML survivors. Staining with the corresponding A *0201/JCV VP1(p100) tetrameric complex showed that VP1(p100)-stimulated peripheral blood mononuclear cells (PBMCs) of 5/7 (71%) PML survivors had JCV-specific CTL, versus none of 6 PML progressors (P = .02). This cellular immune response may therefore be crucial in the prevention of PML disease progression and the tetramer staining assay may be used as a prognostic marker in the clinical management of these patients.     

Major histocompatibility complex molecule expression in the human central nervous system: immunohistochemical analysis of 40 patients

To determine factors affecting major histocompatibility complex (MHC) molecule expression in situ in the human central nervous system (CNS) cryostat tissue sections from 36 autopsies and four biopsies were stained by immunoperoxidase with antibodies to class I (HLA-alpha chain, beta-2 microglobulin), class II (HLA-DR, HLA-DQ) MHC, lymphocyte, and macrophage antigens and Factor VIII-related antigen (VIII-RA). Stained cells and vessels/mm2 were counted in gray and white matter of four CNS anatomic levels. Class I molecules were found on parenchymal and endothelial cells (approximately 50% of VIII-RA + vessels) but not neurons, and were more abundant in gray than white matter (p less than 0.02). Class I molecules were absent in infants, but in adults expression was unaffected by age, sex, postmortem interval, presence of CNS lesions, or systemic illnesses. Expression of HLA-alpha chain and beta-2 microglobulin were the same. Class II molecules were usually absent but were found on parenchymal and endothelial cells in older adults, most frequently in association with macrophage infiltrates and spinal cord tract degenerations, but not with systemic illnesses. Expression of HLA-DR was greater than that of HLA-DQ. In the human CNS, regulation regulation of expression of MHC molecules is complex, can be affected by age, regional anatomy, and by local or remote CNS lesions, and may influence patterns and degrees of T cell immune responses.     

Constitutive and visna virus induced expression of class I and II major histocompatibility complex antigens in the central nervous system of sheep and their role in the pathogenesis of visna lesions

Expression of major histocompatibility complex (MHC) antigens was studied in the brains of 10 healthy sheep 2 months to 5 years old and 13 sheep infected with visna virus by intracerebral inoculation and killed one and 6 months post infection (p.i.). In healthy sheep there was prominent expression of class I, mainly on endothelial cells but also detected on ependyma, choroid plexus and in the leptomeninges. Class II expression was sparse. It was observed on perivascular cells, in choroid plexus, leptomeninges and on microglial cells in the white matter. No definite increase with age in the constitutive expression of class I and II was observed, confirming that we are dealing with a true constitutive expression. In visna-infected sheep a considerable induction of MHC antigens on microglia was observed, which correlated with severity of lesions and was mainly found in or adjacent to inflammatory infiltrates of the white matter. Increase in class II antigen expression was detected in all sheep but class I only in sheep with the most severe lesions 6 months p.i., an indication of a higher threshold for induction of class I than class II antigens on microglia. Few cells expressed viral antigens, indicating that direct immune-mediated destruction of infected cells plays a minor role in evolution of lesions. Since the preferential induction of MHC antigens on microglia in the white matter correlated with the lesion pattern, activated microglia may play a considerable role in the pathogenesis of lesions.     

Implications of progressive multifocal leukoencephalopathy and JC virus for the etiology of MS

JCV infects oligodendrocytes and, to a lesser extent, astrocytes in the brain and spinal cord and causes the demyelinating disease known as progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals. The possibility exists that this opportunistic infection reactivates from a latent state in the brain. It is proposed that the pathogenetic immune response in a multiple sclerosis (MS) brain may be directed predominantly toward antigens of a DNA virus, such as JCV, which is latent in glial cells. The target antigens could be synthesized only during transient viral reactivation or could persist, thus explaining the two basic patterns of neurological symptoms in MS. It is further proposed that the viral genome as a minichromosome becomes focally distributed in glial cells following vertical passage in dividing progenitor cells after infection early in life. The concept that the host response to a single agent can evoke two distinct pathologies (PML and MS) derives from a chronic mycobacterial infection of peripheral nerves-leprosy.     

Immune surveillance and response to JC virus infection and PML

The ubiquitous human polyomavirus JC virus (JCV) is the established etiological agent of the debilitating and often fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Most healthy individuals have been infected with JCV and generate an immune response to the virus, yet remain persistently infected at subclinical levels. The onset of PML is rare in the general population, but has become an increasing concern in immunocompromised patients, where reactivation of JCV leads to uncontrolled replication in the CNS. Understanding viral persistence and the normal immune response to JCV provides insight into the circumstances which could lead to viral resurgence. Further, clues on the potential mechanisms of reactivation may be gleaned from the crosstalk among JCV and HIV-1, as well as the impact of monoclonal antibody therapies used for the treatment of autoimmune disorders, including multiple sclerosis, on the development of PML. In this review, we will discuss what is known about viral persistence and the immune response to JCV replication in immunocompromised individuals to elucidate the deficiencies in viral containment that permit viral reactivation and spread.     

Immunological reactions to neural grafts in the central nervous system

Immunological rejection is a lasting, although highly variable, threat to allo- and xenogeneic neural tissue grafted to the CNS of rodents, monkeys and man. One major determinant for rejection of intracerebral CNS grafts appears to be induction of major histocompatibility complex (MHC) antigens on the donor CNS cells. We have previously examined the cellular immune response against neural mouse xenografts undergoing rejection in the adult rat brain. In this study we focus on the astro- and microglial reactions within and around the graft, and the potential of individual host rat and donor mouse brain cells to express MHC antigens. Previous light microscopical observations of expression of rat MHC antigen class I by endothelial cells, microglial cells, and invading leukocytes were extended to the ultrastructural level and found to include a few astrocytes. Rat and mouse MHC antigen class II was only detected on leukocytes and activated microglial cells. The findings imply that within grafts of brain or spinal cord tissue donor astrocytes, microglial cells and endothelial cells can be induced to act as target cells for class I specific host T cytotoxic cells, while only (graft and host) microglial cells can be induced to express MHC antigen class II and present antigen to sensitized (and possibly also resting) host T helper cells.     

Impaired immune responsiveness is an essential component in persistent central nervous system infection with gross murine leukemia virus

Exposure of newborn mice to Gross murine leukemia virus (GMuLV) results in persistent viral infection of the central nervous system (CNS) white matter. Animals exposed to virus as neonates showed a marked depression in GMuLV-specific B lymphocyte function as evidenced by significant decreases in adult and neonatal anti-GMuLV antibody levels. Immunohistochemical analyses showed that the sites of GMuLV infection in the CNS were also devoid of major histocompatibility complex (MHC) class I and II protein expression, although transplantation of GMuLV-infected brain tissue to the kidney capsules of immunocompetent mice induced a potent mononuclear cell graft infiltrate. These results indicate that persistent GMuLV infection of the CNS is linked to both impairment of anti-GMuLV peripheral immune responses and deficient antigen-presenting cell function within the CNS.     

High expression of MeCP2 in JC virus‐infected cells of progressive multifocal leukoencephalopathy brains

Mutations of the methyl CpG binding protein 2 (MeCP2) gene are a major cause of Rett syndrome. To investigate whether the expression of this gene was related to JC virus (JCV) infection, we examined brains of four progressive multifocal leukoencephalopathy (PML) patients. JCV infection was confirmed by immunohistochemical labeling with antibodies against JCV VP1, agnoprotein and large T antigen. MeCP2 expression was examined by immunohistochemistry using a specific polyclonal antibody against MeCP2. In normal brains and uninfected cortices of PML brains, MeCP2 expression was observed in the nuclei of neurons, but not observed in glial and endothelial cell nuclei. However, in PML brains intense immunolabeling was observed in abnormally enlarged glial nuclei of JCV‐infected cells. Double immunolabeling using antibodies against large T antigen (visualized as blue) and MeCP2 (visualised as red) revealed dark red JCV‐infected nuclei, which confirmed that the JCV infected nuclei expressed MeCP2. We conclude that MeCP2 is highly expressed in the JCV‐infected nuclei of PML brain and these results may provide a new insight into the mechanism which regulates the MeCP2 expression in glial cells by the infection of JCV.     

Early pathological changes in progressive multifocal leukoencephalopathy: a report of two asymptomatic cases occurring prior to the AIDS epidemic

Serial sections of formalin-fixed, paraffinembedded blocks from two asymptomatic, non-AIDS cases of progressive multifocal leukoencephalopathy (PML) were stained with a double-label immunocytochemical method for detection of glial fibrillary acidic protein and JC virus (JCV) capsid proteins and with luxol fast blue/hematoxylin-eosin. In case 1 small, rounded lesions of about 1-mm diameter were seen within a restricted area in the posterior part of the superior frontal gyrus of both cerebral hemispheres, suggesting an early manifestation of the disease. Fully developed demyelinated lesions of the classical type with JCV-infected oligodendrocytes appeared in the white matter and along its border with the cortex. Lesswell-developed lesions, believed to be precursors to the fully developed ones, were seen in the gray and white matter. Of special interest were areas which contained small collections of enlarged, glial fibrillary acidic protein (GFAP)-positive astrocytes without capsid antigen and which seemed to lack destruction of myelin as judged from the appearance of matching serial sections stained for myelin. Large lesions in the brain of case 2 showed the well-known features of advanced PML. The close relation between some astrocytes and oligodendrocytes with viral antigen raises the possibility of early intercellular passage of virus. Vacuolation, seen within or near lesions in both cases, has previously been noted in the CNS infected by HIV, but not in PML. It is suggested that PML, a disease of both oligodendrocytes and astrocytes, may actually begin in astroglial cells which, under the influence of a restricted JCV infection, become reactive, express GFAP and pass on virus to the more highly susceptible oligodendrocytes with which they are in contact.Supported in part by a grant N.S.07596 from the National Institute of Neurological Disorders and Stroke. The work was carried out in the Laboratory of Experimental Neurophathology, NINDS, and in the Department of Pathology II, Karolinska Institute, Stockholm     

Association of human polyomavirus JC with peripheral blood of immunoimpaired and healthy individuals

JC virus (JCV) infection is regularly asymptomatic in healthy individuals. In contrast, in immunocompromised individuals, highly activated virus replication may lead to PML. Peripheral blood cells (PBCs) are found to habor JCV DNA in healthy and diseased individuals and it is discussed that they might be responsible for dissemination of the virus to the central nervous system (CNS) during persistence. To better understand the role of JCV DNA in PBCs for persistent infection and pathogenesis, the authors characterized the extent of JCV infection in Ficoll-gradient purified blood cells (peripheral blood mononuclear cells [PBMCs]) of healthy and human immunodeficiency virus type 1 (HIV-1)-infected individuals. Virus activation in PBMCs from healthy JCV-infected individuals was found at a rate of 0% to 38% at low polymerase chain reaction (PCR) sensitivity. In progressive multifocal leukoencephalopathy (PML) patients, a stronger signal was found, indicating increased virus activation. JCV DNA was regularly detected in T and B lymphocytes and in monocytes at low levels. However, granulocytes were shown to be the predominant reservoir of JCV DNA harboring high copy numbers. Although the overall distribution of viral genomes holds true for the population studied, in the individual, a markedly changed pattern of distribution can be found.     

Association of human polyomavirus JC with peripheral blood of immunoimpaired and healthy individuals

JC virus (JCV) infection is regularly asymptomatic in healthy individuals. In contrast, in immunocompromised individuals, highly activated virus replication may lead to PML. Peripheral blood cells (PBCs) are found to harbor JCV DNA in healthy and diseased individuals and it is discussed that they might be responsible for dissemination of the virus to the central nervous system (CNS) during persistence. To better understand the role of JCV DNA in PBCs for persistent infection and pathogenesis, the authors characterized the extent of JCV infection in Ficoll-gradient purified blood cells (peripheral blood mononuclear cells [PBMCs]) of healthy and human immunodeficiency virus type 1 (HIV-1)-infected individuals. Virus activation in PBMCs from healthy JCV-infected individuals was found at a rate of 0% to 38% at low polymerase chain reaction (PCR) sensitivity. In progressive multifocal leukoencephalopathy (PML) patients, a stronger signal was found, indicating increased virus activation. JCV DNA was regularly detected in T and B lymphocytes and in monocytes at low levels. However, granulocytes were shown to be the predominant reservoir of JCV DNA harboring high copy numbers. Although the overall distribution of viral genomes holds true for the population studied, in the individual, a markedly changed pattern of distribution can be found.     

Herpes simplex virus type I infection of the CNS induces major histocompatibility complex antigen expression on rat microglia

Rats were infected with herpes simplex virus type I (HSV-1) by corneal scarification. The spread of virus in the brain, the infiltration of leucocytes into infected areas, and the expression of major histocompatibility complex (MHC) glycoproteins by brain cells were assessed as a function of time by immunohistochemistry. Virus moved along neuronal pathways, achieving widespread distribution in the brain by days 8-10 when the illness appeared most severe. Granulocytes, T-lymphocytes, and monocytes infiltrated the tissue matrix at sites of infection. Microglial cells were induced to express MHC class I and class II glycoproteins. Reactive microglia near the sites of infection most vigorously expressed such glycoproteins. At the peak of the infection they were detectable on microglia throughout the brain, including areas apparently separated from active infection. Evidence of viral antigens, as well as microglial MHC expression, had largely disappeared by day 30. Neurons, astrocytes, and oligodendroglial cells failed to express MHC antigens.     

The heterogeneity in the immune response and efficiency of viral dissemination in brain infected with herpes simplex virus type 1 through peripheral or central route

Using immunohistochemistry on adjacent brain sections, we studied the correlation between the dissemination of the virus, the inflammatory responses and the expression of major histocompatibility complex (MHC) proteins in rat brain infected with herpes simplex virus (HSV-1) F strain by either corneal scarification or intracerebral injection. Our results showed that the mortality of the corneally infected rats was much higher than that of the intracerebrally infected rats, due to a more extensive dissemination of the virus in the brain, particularly in the brain stem. The inflammatory responses were similar in brains infected through either route, as demonstrated by the expression of MHC I/II antigens on infiltrating lymphocytes, leukocytes and macrophage/microglia cells. While there was strong immunoreactivity for HSV-1 antigens in the cerebral cortex, the infiltrates were only located in subcortical areas, especially the hippocampus. Therefore, the distribution of these immune cells did not always overlap with the regions of viral infection. These results suggest that HSV-1 disseminate more efficiently from the peripheral to the central nervous system (CNS) than from CNS to CNS, which is independent of the immune responses, and that the cerebral cortex may immunologically respond to HSV-1 infection differently from other brain regions. Received: 16 June 1998 / Revised: 22 October 1998 / Accepted: 11 November 1998     

HIV-associated progressive multifocal leukoencephalopathy: longitudinal study of JC virus non-coding control region rearrangements and host immunity

John Cunningham virus (JCV), the etiological agent of progressive multifocal leukoencephalopathy (PML), contains a hyper-variable non-coding control region usually detected in urine of healthy individuals as archetype form and in the brain and cerebrospinal fluid (CSF) of PML patients as rearranged form. We report a case of HIV-related PML with clinical, immunological and virological data longitudinally collected. On admission (t0), after 8-week treatment with a rescue highly active antiretroviral therapy (HAART), the patient showed a CSF-JCV load of 16,732 gEq/ml, undetectable HIV-RNA and an increase of CD4+ cell count. Brain magnetic resonance imaging (MRI) showed PML-compatible lesions without contrast enhancement. We considered PML-immune reconstitution inflammatory syndrome as plausible because of the sudden onset of neurological symptoms after the effective HAART. An experimental JCV treatment with mefloquine and mirtazapine was added to steroid boli. Two weeks later (t1), motor function worsened and MRI showed expanded lesions with cytotoxic oedema. CSF JCV-DNA increased (26,263 gEq/ml) and JCV viremia was detected. After 4 weeks (t2), JCV was detected only in CSF (37,719 gEq/ml), and 8 weeks after admission (t3), JC viral load decreased in CSF and JCV viremia reappeared. The patient showed high level of immune activation both in peripheral blood and CSF. He died 4 weeks later. Considering disease progression, combined therapy failure and immune hyper-activation, we finally classified the case as classical PML. The archetype variant found in CSF at t0/t3 and a rearranged sequence detected at t1/t2 suggest that PML can develop from an archetype virus and that the appearance of rearranged genotypes contribute to faster disease progression.