JC Virus Latency in the Brain and Extraneural Organs of Patients with and without Progressive Multifocal Leukoencephalopathy

JC virus (JCV) is latent in the kidneys and lymphoid organs of healthy individuals, and its reactivation in the context of immunosuppression may lead to progressive multifocal leukoencephalopathy (PML). Whether JCV is present in the brains or other organs of healthy people and in immunosuppressed patients without PML has been a matter of debate. We detected JCV large T DNA by quantitative PCR of archival brain samples of 9/24 (38%) HIV-positive PML patients, 5/18 (28%) HIV-positive individuals, and 5/19 (26%) HIV-negative individuals. In the same samples, we detected JCV regulatory region DNA by nested PCR in 6/19 (32%) HIV-positive PML patients, 2/11 (18%) HIV-positive individuals, and 3/17 (18%) HIV-negative individuals. In addition, JCV DNA was detected in some spleen, lymph node, bone, and kidney samples from the same groups. In situ hybridization data confirmed the presence of JCV DNA in the brains of patients without PML. However, JCV proteins (VP1 or T antigen) were detected mainly in the brains of 23/24 HIV-positive PML patients, in only a few kidney samples of HIV-positive patients, with or without PML, and rarely in the bones of HIV-positive patients with PML. JCV proteins were not detected in the spleen or lymph nodes in any study group. Furthermore, analysis of the JCV regulatory region sequences showed both rearranged and archetype forms in brain and extraneural organs in all three study groups. Regulatory regions contained increased variations of rearrangements correlating with immunosuppression. These results provide evidence of JCV latency in the brain prior to severe immunosuppression and suggest new paradigms in JCV latency, compartmentalization, and reactivation.JC virus (JCV) is the etiologic agent of the often fatal brain-demyelinating disease progressive multifocal leukoencephalopathy (PML) (23a). JCV remains latent in the kidneys, lymph nodes, and bone marrow of healthy and immunosuppressed individuals without PML (2, 21, 24) and, upon reactivation, can cause a lytic infection of oligodendrocytes in the brain, leading to PML (14). Although JCV is often found in the urine of healthy individuals (12, 18), it is not usually detected in the blood of patients without PML (15). The pathway leading to viral reactivation and replication in the brains of immunosuppressed individuals is not well defined. Molecular analysis of JCV has prompted hypotheses on how the virus emerges from latency and becomes pathogenic. JCV has a double-stranded, circular DNA of 5,130 bp. While the coding region is well conserved, the noncoding regulatory region (RR) of JCV is hypervariable. The kidneys and urine usually contain JCV with a well-conserved, nonpathogenic RR which is called the “archetype” (30). The JCV RR detected in the brains and the cerebrospinal fluid (CSF) of PML patients usually has duplications, tandem repeats, and deletions and has been called “rearranged” compared to the archetype. Although it is not clear which form of JCV RR is propagated at the time of primary infection, it has been hypothesized that JCV with the archetype RR remains confined in the kidneys of most healthy individuals and that rearrangements which confer neurotropism need to occur prior to viral migration to the brain to destroy the myelin-producing glial cells. Whether JCV can reach the brain and establish latency in the central nervous systems (CNS) of otherwise-healthy individuals are matters of debate. While some investigators detected JCV DNA in 28 to 68% of frozen (8, 27) and 18 to 71% of formalin-fixed, paraffin-embedded (FFPE) (4, 7, 20) brain samples of patients without PML, others reported negative results (3, 6, 10, 23). Clearly, characterizing JCV sites of latency is imperative in the prevention of viral reactivation and PML. Recently, a group of PML patients has emerged among those treated with monoclonal antibodies, including natalizumab (13, 17, 26), efalizumab (16, 19a), and rituximab (5), for multiple sclerosis, psoriasis, hematological malignancies, and rheumatologic diseases. Mechanisms of JCV reactivation in these patients has yet to be defined. To better understand JCV organ tropism and characterize the types of JCV RRs in different compartments, we used archival pathology samples to detect JCV DNA and proteins and to analyze JCV RRs in various organ systems in HIV-positive individuals with and without PML and in HIV-negative subjects.     

JC virus-specific cytotoxic T lymphocytes in individuals with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by a reactivation of the polyomavirus JC (JCV) within a setting of immunosuppression. The nature of the immune response that contains replication of this virus is unknown. We have explored JCV-specific cellular immune responses in patients with PML and control subjects. JCV antigen-stimulated peripheral blood mononuclear cells (PBMC) of four human immunodeficiency virus (HIV)-infected patients who were survivors of PML and one HIV-uninfected patient recently diagnosed with PML lysed autologous B-lymphoblastoid cell lines expressing either the JCV T regulatory protein or the VP1 major capsid protein. This lysis was mediated by CD8(+) T lymphocytes and was major histocompatibility complex class I restricted. These cells were therefore cytotoxic T lymphocytes (CTL). JCV-specific CTL could not be detected in PBMC of three HIV-infected PML patients who had progressive neurologic disease and an eventual fatal outcome. These data suggest that the JCV-specific cellular immune response may play a crucial role in the containment of PML. This finding may also prove useful as a favorable prognostic marker in the clinical management of these patients.     

Frequency and phenotype of JC virus-specific CD8+ T lymphocytes in the peripheral blood of patients with progressive multifocal leukoencephalopathy

JC virus (JCV)-specific CD8+ cytotoxic T lymphocytes (CTL) are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML) and cross-recognize the polyomavirus BK virus (BKV). We sought to determine the frequency and phenotype in fresh blood of CD8+ T cells specific for two A*0201-restricted JCV epitopes, VP1(p36) and VP1(p100), and assess their impact on JC and BK viremia and viruria in 15 healthy subjects, eight human immunodeficiency virus-positive (HIV+) individuals, and nine HIV+ patients with PML (HIV+ PML patients) classified as survivors. After magnetic pre-enrichment of CD8+ T cells, epitope-specific cells ranged from 0.001% to 0.022% [corrected] by tetramer staining, with no significant difference among the three study groups. By use of seven-color flow cytometry, there was no predominant differentiation phenotype subset among JCV-specific CD8+ T cells in healthy individuals, HIV+ subjects, or HIV+ PML patients. However, in one HIV+ PML patient studied in the acute phase, there was a majority of activated effector memory cells. BKV DNA was undetectable in all blood samples by quantitative PCR, while a low JC viral load was found in the blood of only one HIV+ and two HIV+ PML patients. JCV and BKV DNA were detected in 33.3% and 13.3% of all urine samples, respectively, independent of the presence of JCV-specific CTL. The detection of JCV DNA in the urine was associated with the presence of a JCV VP1(p100) CTL response. Immunotherapies aiming at increasing the cellular immune response against JCV may be valuable in the treatment of HIV+ individuals with PML.     

CRISPR/Cas9 System as an Agent for Eliminating Polyomavirus JC Infection

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by reactivation of the human polyomavirus JCV gene expression and its replication in oligodendrocytes, the myelin producing cells in the brain. Once a rare disease seen in patients with lymphotproliferative and myeloproliferative disorders, PML has been seen more frequently in HIV-1 positive/AIDS patients as well as patients undergoing immunomodulatory therapy due for autoimmune disorders including multiple sclerosis, rheumatoid arthritis, and others. As of now there is no cure for PML and in most cases disease progression leads to death within two years. Similar to other polyomaviruses, the JCV genome is small circular double stranded DNA that includes coding sequences for the viral early protein, T-antigen, which is critical for directing viral reactivation and lytic infection. Here, we employ a newly developed gene editing strategy, CRISPR/Cas9, to introduce mutations in the viral genome and, by inactivating the gene encoding T-antigen, inhibit viral replication. We first used bioinformatics screening and identified several potential targets within the JCV T-antigen gene that can serve as sites for the creation of guide RNAs (gRNAs) for positioning the Cas9 nuclease on the designated area of the viral genome for editing. Results from a series of integrated genetic and functional studies showed that transient or conditional expression of Cas9 and gRNAs specifically targets the DNA sequences corresponding to the N-terminal region of T-antigen, and by introducing mutation, interferes with expression and function of of the viral protein, hence suppressing viral replication in permissive cells. Results from SURVEYOR assay revealed no off-target effects of the JCV-specific CRISPR/Cas9 editing apparatus. These observations provide the first evidence for the employment of a gene editing strategy as a promising tool for the elimination of the JCV genome and a potential cure for PML.     

Changes in JC Virus-Specific T Cell Responses during Natalizumab Treatment and in Natalizumab-Associated Progressive Multifocal Leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) induced by JC virus (JCV) is a risk for natalizumab-treated multiple sclerosis (MS) patients. Here we characterize the JCV-specific T cell responses in healthy donors and natalizumab-treated MS patients to reveal functional differences that may account for the development of natalizumab-associated PML. CD4 and CD8 T cell responses specific for all JCV proteins were readily identified in MS patients and healthy volunteers. The magnitude and quality of responses to JCV and cytomegalovirus (CMV) did not change from baseline through several months of natalizumab therapy. However, the frequency of T cells producing IL-10 upon mitogenic stimulation transiently increased after the first dose. In addition, MS patients with natalizumab-associated PML were distinguished from all other subjects in that they either had no detectable JCV-specific T cell response or had JCV-specific CD4 T cell responses uniquely dominated by IL-10 production. Additionally, IL-10 levels were higher in the CSF of individuals with recently diagnosed PML. Thus, natalizumab-treated MS patients with PML have absent or aberrant JCV-specific T cell responses compared with non-PML patients, and changes in T cell-mediated control of JCV replication may be a risk factor for developing PML. Our data suggest further approaches to improved monitoring, treatment and prevention of PML in natalizumab-treated patients.     

Establishment of COS‐JC cells persistently producing archetype JC polyomavirus

JC polyomavirus (JCPyV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunocompromised patients. Archetype JCPyV circulates in the human population. There have been several reports of archetype JCPyV replication in cultured cells, in which propagation was not enough to produce high titers of archetype JCPyV. In this study, we carried out cultivation of the transfected cells with archetype JCPyV DNA MY for more than 2 months to establish COS‐7 cells (designated COS‐JC cells) persistently producing archetype JCPyV. Moreover, JCPyV derived from COS‐JC cells was characterized by analyzing the viral propagation, size of the viral genome, amount of viral DNA, production of viral protein, and structure of the non‐coding control region (NCCR). Southern blotting using a digoxigenin‐labeled JCPyV probe showed two different sizes of the JCPyV genome in COS‐JC cells. For molecular cloning, four of five clones showed a decrease in the size of complete JCPyV genome. Especially, clone No. 10 was generated the large deletion within the Large T antigen. On the other hand, the archetype structure of the NCCR was maintained in COS‐JC cells, although a few point mutations occurred. Quantitative PCR analysis of viral DNA in COS‐JC cells indicated that a high copy number of archetype JCPyV DNA was replicated in COS‐JC cells. These findings suggest that COS‐JC cells could efficiently propagate archetype JCPyV MY and offer a useful tool to study persistent infection of archetype JCPyV in a kidney‐derived system.

     

Molecular biology and immunoregulation of human neurotropic JC virus in CNS

The human polyomavirus, JC virus (JCV), provides an excellent model system to investigate the reciprocal interaction of the immune and nervous systems. Infection with JCV occurs during childhood and the virus remains in the latent state with no apparent clinical symptoms. However, under immunosuppressed conditions, the virus enters the lytic cycle and upon cytolytic destruction of glial cells, causes the fatal demyelinating disease of the central nervous system (CNS), named progressive multifocal leukoencephalopathy (PML). In this short review, we discuss the molecular pathogenesis of PML by highlighting the role of the immune system in modulating JCV gene activation and replication, and the latency/reactivation of this virus upon immunosuppression. Further, due to the higher incidence of PML among AIDS patients, we further elaborate on the cross-talk between JCV and HIV-1 by direct and indirect pathways that lead to enhanced expression of the JCV genome.     

Detection of JC virus-specific cytotoxic T lymphocytes in healthy individuals

The polyomavirus JC (JCV) infects 85% of healthy individuals, and its reactivation in a limited number of immunosuppressed people causes progressive multifocal leukoencephalopathy (PML), a severe demyelinating disease of the central nervous system. We hypothesized that JCV-specific cytotoxic T lymphocytes (CTLs) might control JCV replication in healthy individuals, blocking the evolution of PML. Using 51Cr release and tetramer staining assays, we show that 8 of 11 HLA-A*0201+ healthy subjects (73%) harbor detectable JCV-specific CD8+ CTLs that recognize one or two epitopes of JCV VP1 protein, the HLA-A*0201-restricted VP1p36 and VPp1100 epitopes. We determined that the frequency of JCV VP1 epitope-specific CTLs varied from less than 1/100,000 to 1/2,494 peripheral blood mononuclear cells. More individuals had JCV VP1-specific than cytomegalovirus-specific CTLs (8 of 11 subjects [73%] versus 2 of 10 subjects [20%], respectively). These results show that a CD8+-T-cell response against JCV is commonly found in immunocompetent people and suggest that these cells might protect against the development of PML.     

Rearranged JC Virus Noncoding Control Regions Found in Progressive Multifocal Leukoencephalopathy Patient Samples Increase Virus Early Gene Expression and Replication Rate

Polyomavirus JC (JCV) infects ∼60% of the general population, followed by asymptomatic urinary shedding in ∼20%. In patients with pronounced immunodeficiency, including HIV/AIDS, JCV can cause progressive multifocal leukoencephalopathy (PML), a devastating brain disease of high mortality. While JCV in the urine of healthy people has a linear noncoding control region called the archetype NCCR (at-NCCR), JCV in brain and cerebrospinal fluid (CSF) of PML patients bear rearranged NCCRs (rr-NCCRs). Although JCV NCCR rearrangements are deemed pathognomonic for PML, their role as a viral determinant is unclear. We sequenced JCV NCCRs found in CSF of eight HIV/AIDS patients newly diagnosed with PML and analyzed their effect on early and late gene expression using a bidirectional reporter vector recapitulating the circular polyomavirus early and late gene organization. The rr-NCCR sequences were highly diverse, but all increased viral early reporter gene expression in progenitor-derived astrocytes, glia-derived cells, and human kidney compared to the expression levels with the at-NCCR. The expression of simian virus 40 (SV40) large T antigen or HIV Tat expression in trans was associated with a strong increase of at-NCCR-controlled early gene expression, while rr-NCCRs were less responsive. The insertion of rr-NCCRs into the JCV genome backbone revealed higher viral replication rates for rr-NCCR compared to those of the at-NCCR JCV in human progenitor-derived astrocytes or glia cells, which was abrogated in SV40 large T-expressing COS-7 cells. We conclude that naturally occurring JCV rr-NCCR variants from PML patients confer increased early gene expression and higher replication rates compared to those of at-NCCR JCV and thereby increase cytopathology.Polyomavirus JC (JCV) infects approximately 60% of the general population, followed by asymptomatic urinary shedding in 20% of healthy individuals (20). Although JCV-associated nephropathy may occur in kidney transplant (14, 33) and HIV/AIDS patients (6, 27), the most prominent JCV disease is progressive multifocal leukoencephalopathy (PML) (44, 60). The pathology of PML was first described in 1958 as a rare complication of patients with chronic lymphocytic leukemia or Hodgkin''s lymphoma (3). Today, PML is recognized as a rare, virus-mediated demyelinating disease of the white brain matter in highly immunocompromised patients, including HIV/AIDS, transplantation, and chemotherapy patients and those exposed to immunomodulatory or depleting biologicals for the treatment of autoimmune diseases (29, 40). During the human immunodeficiency virus type 1 (HIV-1) pandemic, the incidence of PML rose significantly to rates of 1 to 8% prior to the use of highly active antiretroviral therapy (2, 5, 34). The definitive diagnosis requires brain tissue, but the detection of JCV by PCR in cerebrospinal fluid (CSF) is generally accepted for a laboratory-confirmed diagnosis in immunocompromised patients with (multi-)focal neurological deficits and corresponding radiological findings (8, 26). Due to the lack of effective antiviral therapy (13), the treatment of PML is based on improving overall immune functions. While this is difficult to achieve in cancer, chemotherapy, and transplantation, prompt antiretroviral therapy in HIV/AIDS patients has significantly improved PML survival, with increasing JCV-specific immune responses and declining intracerebral JCV replication (7, 15, 23, 35, 37). In patients diagnosed with PML after treatment with natalizumab for multiple sclerosis or inflammatory bowel disease, the removal of the monoclonal antibody by plasmapheresis has been tried to restore lymphocyte homing to, and the immune surveillance of, JCV replication sites in the central nervous system (38, 40, 52). However, the success of immune reconstitution in HIV/AIDS- and natalizumab-associated PML cases is limited by the fact that PML is typically diagnosed clinically by neurological deficits resulting from significant brain damage, where mounting antiviral immunity often may be too slow to modify the outcome. On the other hand, rapid recovery may cause immune reconstitution inflammatory syndrome with paradoxical clinical worsening and fatal outcomes (9, 16, 25, 38, 46). Although the etiologic role of JCV in PML is well documented, the pathogenesis and, in particular, the role of viral determinants is less clear. Virtually all JCV strains isolated from the brain or CSF of PML patients are characterized by highly variable genomic rearrangements of the noncoding control region (NCCR), which governs viral early and late genes in opposite directions of the circular polyomavirus DNA genome (1, 4, 31, 39, 41, 43, 49, 54, 59). In contrast, JCV detected in the urine of immunocompetent individuals show a consistent linear architecture called the archetype NCCR (at-NCCR). Thus, detecting rearranged NCCRs (rr-NCCRs) JCV in the central nervous system has been viewed as being derived from the archetype and closely linked to PML (4), but the functional consequences of rearrangements are unclear. To address the consequences of the rr-NCCR for JCV gene expression and replication, we characterized the sequences of JCV rr-NCCR from patients with PML and analyzed their effect on viral gene expression and replication with JCV at-NCCR in a bidirectional reporter assay and in recombinant JCV.     

The pathological role of Wnt5a in psoriasis and psoriatic arthritis

Psoriasis (PsO) is a chronic inflammatory skin disease with both local and systemic components. PsO‐associated arthritis, known as psoriatic arthritis (PsA), develops in approximately 13%‐25% of PsO patients. Various factors associated with both PsO and PsA indicate that these conditions are part of a single disease. Identification of novel targets for the development of drugs to treat both PsO and PsA is desirable to provide more patient‐friendly treatment regimens. Such targets will likely represent ‘common checkpoints’ of inflammation, for example key components or transduction cascades of the signalling pathways involved. Emerging evidence supports involvement of the non‐canonical Wnt signalling pathways in the development of both PsO and PsA, especially the Wnt5a‐activated signalling cascades. These, together with interlinked factors, are crucial in the interactions among keratinocytes, immune cells and inflammatory factors in PsO, as well as among chondrocytes, osteoblasts and osteoclasts that trigger both subchondral bone remodelling and cartilage catabolism in PsA. This review focuses on the pathological role of Wnt5a signalling and its interaction with other interlinked pathways in both PsO and PsA, and also on the main challenges for future research, particularly with respect to molecules targeting Wnt signalling pathways for the treatment of PsO and PsA.     

Fine Mapping Major Histocompatibility Complex Associations in Psoriasis and Its Clinical Subtypes

Psoriasis vulgaris (PsV) risk is strongly associated with variation within the major histocompatibility complex (MHC) region, but its genetic architecture has yet to be fully elucidated. Here, we conducted a large-scale fine-mapping study of PsV risk in the MHC region in 9,247 PsV-affected individuals and 13,589 controls of European descent by imputing class I and II human leukocyte antigen (HLA) genes from SNP genotype data. In addition, we imputed sequence variants for MICA, an MHC HLA-like gene that has been associated with PsV, to evaluate association at that locus as well. We observed that HLA-C^∗06:02 demonstrated the lowest p value for overall PsV risk (p = 1.7 × 10⁻³⁶⁴). Stepwise analysis revealed multiple HLA-C^∗06:02-independent risk variants in both class I and class II HLA genes for PsV susceptibility (HLA-C^∗12:03, HLA-B amino acid positions 67 and 9, HLA-A amino acid position 95, and HLA-DQα1 amino acid position 53; p < 5.0 × 10⁻⁸), but no apparent risk conferred by MICA. We further evaluated risk of two major clinical subtypes of PsV, psoriatic arthritis (PsA; n = 3,038) and cutaneous psoriasis (PsC; n = 3,098). We found that risk heterogeneity between PsA and PsC might be driven by HLA-B amino acid position 45 (p_omnibus = 2.2 × 10⁻¹¹), indicating that different genetic factors underlie the overall risk of PsV and the risk of specific PsV subphenotypes. Our study illustrates the value of high-resolution HLA and MICA imputation for fine mapping causal variants in the MHC.     

JC virus infection of hematopoietic progenitor cells, primary B lymphocytes, and tonsillar stromal cells: implications for viral latency.

The human polyomavirus JC virus (JCV) infects myelin-producing cells in the central nervous system, resulting in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV-induced PML occurs most frequently in immunosuppressed individuals, with the highest incidence in human immunodeficiency type 1-infected patients, ranging between 4 and 6% of all AIDS cases. Although JCV targets a highly specialized cell in the central nervous system, infection is widespread, with more than 80% of the human population worldwide demonstrating serum antibodies. A number of clinical and laboratory studies have now linked the pathogenesis of PML with JCV infection in lymphoid cells. For example, JCV-infected lymphocytes have been suggested as possible carriers of virus to the brain following reactivation of a latent infection in lymphoid tissues. To further define the cellular tropism associated with JCV, we have attempted to infect immune system cells, including CD34+ hematopoietic progenitor cells derived from human fetal liver, primary human B lymphocytes, and human tonsillar stromal cells. Our results demonstrate that these cell types as well as a CD34+ human cell line, KG-1a, are susceptible to JCV infection. JCV cannot, however, infect KG-1, a CD34+ cell line which differentiates into a macrophage-like cell when treated with phorbol esters. In addition, peripheral blood B lymphocytes isolated by flow cytometry from a PML patient demonstrate JCV infection. These results provide direct evidence that JCV is not strictly neurotropic but can infect CD34+ hematopoietic progenitor cells and those cells which have differentiated into a lymphocytic, but not monocytic, lineage.     

Efficient propagation of progressive multifocal leukoencephalopathy-type JC virus in COS-7-derived cell lines stably expressing Tat protein of human immunodeficiency virus type 1

The high incidence of progressive multifocal leukoencephalopathy (PML) in AIDS patients compared with many other immunosuppressive diseases suggests that HIV-1 infection is strictly related to the activation of JC virus (JCV) propagation. In this report, propagation of PML-type JCV in COS-7-derived cell lines stably expressing HIV-1 Tat (COS-tat cells) has been examined. In COS-tat cells, production of viral particles and replication of genomic DNA were markedly increased compared to COS-7 cells, as judged by HA and real-time PCR analyses. These results demonstrate that COS-tat cells provide a useful model system for studying HIV-1 Tat-mediated propagation of PML-type JCV.     

JC virus DNA is present in many human brain samples from patients without progressive multifocal leukoencephalopathy.

Sections of normal and diseased brain and kidney tissues were screened for the presence of JC virus (JCV) DNA by using the polymerase chain reaction. As expected, all samples obtained from patients with progressive multifocal leukoencephalopathy (PML) tested positive when multiple JCV-specific primer and probe combinations were used. Unexpectedly, more than 50% of non-PML-affected brains were also found to harbor low levels of JCV DNA. To confirm that the positive signals seen in the tissue sections were not the result of contamination, amplified DNA was cloned and sequenced and in some cases was shown to represent strains of JCV not identified previously. Two predominant regulatory region configurations of JCV have been detected in the human host: archetype JCV, which is excreted in the urine of normal and immunocompromised individuals, and "PML-type" JCV found in diseased brains. This latter group of variants appears to derive from archetype JCV by the deletion and duplication of sequences within the promoter-enhancer region. In the present study, the archetype strain of JCV was identified only in normal kidney samples; JCV DNA found in non-PML-affected brain specimens and in kidney tissue from patients with PML resembled that of strains isolated from PML-affected brain tissue. Our findings indicate that JCV reaches the brain more frequently than previously thought and may persist at this site without causing demyelinating disease. A subsequent episode of prolonged immunodeficiency or a direct interaction with an immunocompromising agent (e.g., human immunodeficiency virus type 1) might activate the latent JCV infection and lead to the development of PML.