Involvement Of Human Viruses Associated With Chronic Neurologic Disease

National Institute Of Neurological Disorders And Stroke

Investigators

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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 developed ELISPOT assays for the detection of virus-specific immune responses to a variety of neurotropic viruses including EBV, CMV and HHV6. We have shown that immunodominant peptides of EBV induce T cell responses to unique regions of EBV in patients with active MS in comparison to healthy controls or MS patients with stable disease. In addition, we have shown that purified CD19+ B cells from active MS patients have a higher frequency of EBV detection than in stable MS patients or control cohorts. These results support a role for EBV as a potential trigger in this disease. We are currently using small molecule inhibitors to interfere with EBV replication in B cells and PBMC of patients with MS. 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. 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 and can demonstrate EBV-specific CD4+ and CD8+ T cells that can be detected by HLA-restricted tetramers. In addition, we are using our newly developed digital based PCR methodology (ddPCR) for the detection of human viruses. We have characterized PCR primer and pair probes for the detection of HTLV-I, HTLV-II, EBV, JCV, CMV and HHV6 A and HHV6B. We have multiplexed this system in which we can now simultaneously detect and quantify herepesvirus sequences in human material. We have also developed this assay for the detection of SarsCOV2 and will explore the extent and magnitude of this virus in recovering COVID19 patients with neurologic signs and symptoms. We continue to extend our work on the detection of the human herpesvirus (HHV-6) from brain resections of patients with mesial temporal lobe epilepsy, patients with neurologic complications following allogeneic bone marrow transplants, and patients with astrocytomas. We have been exploring the clinical consequences of inherited chromosomally integrated HHV-6 (iciHHV-6) which affects approximately 1% of the human population. ciHHV6 integrates into host chromosomes at one copy of HHV-6 DNA per cell while it remains unclear if HHV-6 reactivates from this integrated state. T cell lines have been generated from iciHHV-6B PBMC and will be tested for reactivation (by a variety of compounds know to stimulate HHV6) by viral expression using PCR assays targeting HHV-6B DNA or RNA, and RNAscope for in situ hybridization. We can induce iciHHV-6 lymphocytes to express HHV-6 RNA in the absence of an increasing viral DNA load. Recently, there has been a re-kindled interest in the infectious-trigger hypothesis of Alzheimers disease (AD) based on bioinformatic analysis of large cohorts of AD autopsy brains compared to controls in which 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 AD and control brain samples. Although we were not able to amplify HHV-6 or other candidate viruses from AD patient samples, this did not preclude a possible association with an early triggering event, rather than disease progression, or these agents may be present at copy numbers below the limit of laboratory detection.