Involvement Of Human Viruses Associated With Chronic Neurologic Disease

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications & trials

Abstract

Major findings from our work have focused on the role of immunopathogenic HTLV-I specific CD8+ cells in the pathology of patients with HAM/TSP. We have shown that the frequency of these cells are elevated in the peripheral blood and even higher in the CSF of HAM/TSP and are directly proportional to the amount of HTLV-I proviral DNA and RNA. Based on the development of novel MRI imaging techniques that can quantify spinal cord atrophy in patients with chronic myelopathies, we have correlated the levels of cytotoxic CD8+ T cells with the extent of spinal cord atrophy in HAM/TSP patients. We have extended the cross-sectional spinal cord analyses to other neurological diseases including multiple sclerosis (MS). We are demonstrating a heterogeneity of atrophic patterns in relapsing-remitting MS. We have also begun to analyze brain atrophy using a novel quantitative atlas-free traditional machine learning method, Classification using Derivative-based Features (C-DEF) for whole brain segmentation, in the setting of limited training data. We will compare observations on spinal cord atrophy with whole brain atrophy and determine immunological correlates as defined by multimodal flow cytometry of cells in the cerebrospinal fluid. Our analysis of T cell receptor repertoires in the peripheral blood and CSF of HAM/TSP and MS patients and have demonstrated T cell clonal expansions particularly in the CSF of MS patients. We have also begun to explore both the T and B cell receptor repertoires from single cells in the CSF of these patients and have successfully expressed paired Vh and Vl chains. The specificity of these antibodies is being investigated. In addition, we have begun single cells RNAseq analysis of PBC and CSF to define the TCR and BCR repertoire of patients with chronic neurologic diseases including MS, HAM/TSP and long-term post COVID-19 patients with neurologic sequelae. One in 4 people are estimated to be infected with SARS-CoV-2 worldwide. Of these, 10% have experienced ongoing symptoms that can last months to years that can include ongoing neurologic issues such as brain fog, anxiety, depression, sleep problems, and headaches, even though the viral RNA become undetectable. It is not yet known how the infection leads to these persistent neurologic symptoms, nor what are the molecular and immunologic mechanisms associated with these conditions. Therefore, we aim to characterize the transcriptome of peripheral blood mononuclear cells (PBMC) and the paired cells isolated from cerebrospinal fluid (CSF) of individuals with neurologic postacute sequelae of SARS-CoV-2 infection (neuro-PASC) using 10X Genomics single cell RNA (scRNA) sequencing. Our preliminary results demonstrate compartmentalized immune profiles can be observed in CSF of individuals with neuro-PASC that was not observed in peripheral blood. We have also been nvestigating whether patterns of antigen-specific antibody responses associated with various viral exposures may define patients with CNS chronic immune dysregulation. Utilizing a pan-viral antibody profiling platform (Virscan) using cerebrospinal fluid and serum of patients with MS, we have demonstrated significant differences from those in healthy volunteers and a pattern of antibody responses against multiple viruses, including Epstein-Barr virus. These findings demonstrate that virus-specific antibody signatures might be able to reflect disease-associated inflammatory milieu in CSF of subjects with neuroinflammatory diseases. We have examined the role of exosomes and extracellular vesicles (EVs) from blood and CSF of patients with known virus-associated neurologic disease including HAM/TSP (HTLV-I) and PML (JC virus). Using multi-parameter immunoflow technology, we have previously characterized these vesicles and determined their cargo for the presence of viral proteins and viral RNAs. Recently, we have analyzed the EVs from the CSF of healthy volunteers and patients with a variety of chronic neurologic diseases of both known viral and non-viral etiologies including MS, HAM/TSP, HTLV-1-infected asymptomatic carriers, and other neurological diseases (ONDs), to investigate the surface repertoires of CSF EVs during disease. Significant increases in CD8+ and CD2+ EVs were found in HAM/TSP patient CSF samples compared to other clinical groups, consistent with the immunopathologically-mediated disease associated with CD8+ cells in the CNS of HAM/TSP patients. Furthermore, CD8+, CD2+, CD44+, and CD40+ EVs were significantly increased in the CSF from patients with viral infections compared to those without. These data suggest that CD8+ and CD2+ CSF EVs may be important as: 1) potential biomarkers for viral-mediated neurological diseases, and 2) as possible meditators of areas of the disease process in infected individuals. Additionally, we have made significant advances in the analysis pipeline for analyzing the concentration of EVs and extracellular particles in biofluids including CSF through microfluidic resistive pulse sensing (MRPS). We can now analyze and compare the concentrations of EVs between disease groups with great reproducibility and accuracy. The purpose of these ongoing studies is to determine if at times when no virus can be detected, EVs particularly from the CSF will carry a viral signature or pattern that can be discerned. We are continuing to assess virus-specific immune responses from MS patients and controls using virus-peptide pools. Although 90% of adults are infected with EBV, recent studies have rekindled a role for this ubiquitous agent in the pathogenesis of MS. To assess whether MS is associated with defective T cell control of EBV-infected B cells, we employed a nanoparticle (np) based Artificial Immune Modulation (AIM) platform consisting of a cocktail of six EBV peptides presented on np decorated with HLA-A2 and anti-CD28 molecules to enrich, expand, and characterize EBV specific CD8+ T cells. The expanded CD8+ T cells showed significant antigen specificity as shown by intracellular cytokine release and/or cytotoxic activity. Interestingly, compared to heathy donors, such cells from MS patients exhibited functional defects in their responses to select EBV peptides. These results, analyzing the functional responses of EBV specific CD8 + T cells expanded from healthy donors and patients with MS, represent our initial effort to interrogate the hypothesis that MS may be associated with defective T cell control of EBV infected cells, which is consistent with reports suggesting that a dysregulation of EBV specific immune responses is associated with the pathogenesis of MS. Recently, there has been interest in the infectious-trigger hypothesis of chronic, inflammatory neurologic diseases including Alzheimers disease (AD) based on bioinformatic analysis of large cohorts of AD autopsy brains compared to controls in which the ubiquitous human bert-herpesvirus,HHV6, was suggested to be associated with AD. This coupled with the observation that extracellular beta-amyloid plaques (a pathologic hallmark of AD) may function as an innate antimicrobial peptide that is released to entrap pathogens and protect the brain from infection, has suggested that Abeta can be nucleated and seeded by microbes in vitro, causing aggregation and subsequent plaque formation. We have performed a bioinformatics and PCR based analysis, on large cohort of brain samples including frontal temporal dementia, Lewey body dementia, AD, and control brain samples (over 6000 brains). Although we were not able to amplify full length HHV-6 or other candidate viruses from these samples, partial sequences particularly for HHV6 were over-represented in patients with dementia than from non-disease controls. These observations support the hypothesis that virus triggers maybe associated with chronic neurologic disease.