Translational studies of human viruses and chronic neurologic disease

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications & trials

Abstract

HTLV-1 was the first defined human pathogenic retrovirus. Seroepidemiologic and molecular genetic data firmly establish an association between HTLV-1 infection and a chronic, slowly progressive neurologic condition previously termed tropical spastic paraparesis (TSP) that often emanated from clusters in tropical areas, including the Caribbean Basin, India, and Africa. A similar condition described in Japan is referred to as HTLV-1-associated myelopathy (HAM). It is now recognized that HAM and TSP (HAM/TSP) are identical clinical conditions. The clinical course in HAM/TSP is predominantly that of a slowly progressive spastic paraparesis associated with bladder and bowel dysfunction. Importantly, this disease shares many clinical similarities with the primary progressive form of multiple sclerosis (MS). Indeed, many HAM/TSP patients have been diagnosed as MS up until the time that HTLV-I has been detected in sera or cerebrospinal fluid (CSF). We examined the cellular immunophenotypes in CSF of patients with HAM/TSP, compared to healthy volunteers and the other virus-associated neurologic diseases including HIV adequately treated with antiretroviral drugs, multiple sclerosis (MS) and progressive multifocal leukoencephalopathy (PML) by multicolor flow cytometry. In patients with HAM/TSP, activated CD4+ T cells were significantly increased in the CSF as well as the blood and correlated with HTLV-1 proviral loads. Increase of activated CD4+ T cells in CSF was also correlated with immunological changes of B cells such as increases of B cell frequency and antibody secreting B cells (ASCs) in CSF of patients with HAM/TSP. Interestingly, CD8+ T cells were increased in the CSF of HAM/TSP patients, which was significantly correlated with spinal cord atrophy. Thus, immunophenotyping of CSF cells may reflect immune pathology in inflammatory neurologic diseases and can serve to highlight differential diagnoses that may lead to a better understanding of disease pathogenesis and clinical treatment. As HAM/TSP is a chronic progressive inflammatory neurological disorder associated with virus-infected circulating T-cells that infiltrate the CNS, we have analyzed T-cell receptor (TCR) repertoire signature in the peripheral blood and CSF of HAM/TSP patients and healthy controls (HC). We have shown that the use of molecular bar codes can lead to more accurate amplification and analysis of the T-cell clonal expansion profile without loss of real repertoire diversity. When we evaluated the TCR repertoire expansion by the frequency of each individual clone a significantly higher clonal expansion was also found in HAM/TSP patients. Of note, relatedness among clones was observed in HAM/TSP patients by phylogenetic tree analysis. We have shown that TCR repertoires of HAM/TSP patients are highly expanded in CSF and contains both TCR clonotypes shared with PBMCs and uniquely enriched clonotypes within the CSF. Analysis of TCR repertoire of HTLV-I Tax-specific CD8+ T cells demonstrated the use of private TCR sequences for the recognition of antigen and identified conserved motifs in the CDR3 region. Moreover, TCR clonotypes of expanded clones in HTLV-1 specific CD8+ T cells in the periphery were also expanded and enriched in the CSF. Exploring TCR repertoire of CSF and antigen-specific T cells may provide a TCR repertoire signature in virus-associated neurologic disorders. We will extend these studies to larger cohorts and include longitudinal time points to see if the TCR repertoire changes with time, treatment, disease progression or with laboratory parameters including HTLV-I virus load and immunological T and B cell subsets. More recently, we have employed single cell RNA sequencing technology (10X genomics) for the transciptomic analysis of blood and CSF of patients with neurologic diseases associated with viruses (HAM/TSP and long Covid) compared to heathy controls. We have measured the spinal cord cross sectional area (SSCSA) using 225 MRI scans from 200 patients with chronic progressive neurological diseases and controls. Building from our previous experience, we have applied this technique to spinal cord atrophy measurements and have characterized the spinal cord neurodegenerative process in HAM/TSP. We could show that SSCA profiles in HAM/TSP correlated with clinical outcome measures of disability including the EDSS and IPEC. Importantly, HAM/TSP SSCA profiles correlated with laboratory-defined immune parameters. Analysis of CSF immunophenotypes demonstrated statistically significant associations between the T4 to T9 subregion of the spinal cord and the percentage of CD8+T cells detected in the CSF. This indicates that a more atrophic cord is associated with a higher percentage of inflammatory CD8+T cells in the CSF, consistent with a role for these cells in HAM/TSP pathogenesis. In addition, we have applied this technique to spinal cord atrophy measurements of MS patients and have shown significant reduction in spinal cord volume particularly at the C1-C4 level. We have initiated a novel whole brain volume measurement using cDEF and will compare spinal cord atrophy with brain atrophy. We have initiated a new project to enrich viruses in cerebrospinal fluid (CSF) samples using a carbon nanotube platform (VIRRION) that captures viruses, coupled to Raman spectroscopy for a rapid identification of viruses in clinical samples. We aim to capture and record Raman spectroscopy fingerprints of JCV, EBV, HHV6, and HTLV-1 captured on the VIRRION carbon nanotube platform and use an established machine learning strategy to classify these results. Nucleic acid will be extracted from the carbon nanotubes for identification using PCR and sequencing. Over time, we will include many more viruses of interest in neurologic disease to establish a catalog of Raman signatures that can be used for identification of viruses from CSF of patients with neurologic disease in which viruses are suspected to play a role. We are optimizing the raman spectra of the virus drop-casted onto a forest of carbon nanotubes (CNTs) and determined the combination of batch size, magnification, and accumulation time that would result in the shortest collection time without reducing accuracy. We constructed convolutional neural networks (CNNs), which are a form of deep learning, to differentiate cell culture samples containing JC virus from blank culture. In addition, we have expanded our studies on the role of EBV in the pathogenesis of MS. We have established spontaneous lymphoblastoid cell lines (SLCLs) generated ex vivo with the endogenous EBV of patients with MS and controls treated with either an Epstein-Barr virus nuclear antigen 1 (EBNA1) inhibitor (VK-1727) or cladribine, a nucleoside analog that eliminates B cells. We showed that this small molecule inhibitor of EBNA1, a critical regulator of the EBV life cycle, blocks the proliferation and metabolic activity of these SLCLs. In contrast to cladribine, a highly cytotoxic B cell depleting therapy currently used in MS, the EBNA1 inhibitor VK-1727 was cytostatic rather than cytotoxic and selective for EBV+ cells, while having no discernible effectson EBV negative cells. We validated that VK-1727 reduces EBNA1 DNA binding at known viral and cellular sites by ChIP-qPCR. These results shows that patient-d