Translational studies of human viruses and chronic neurologic disease

National Institute Of Neurological Disorders And Stroke

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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). A major emphasis within the Viral Immunology Section is to examine clinical, virological and immunological parameters in individuals with HAM/TSP, asymptomatic sero-positive individuals, family members at risk for acquiring HTLV-1, and individuals with HTLV-1 indeterminate serology. Specific focus is on the effect of viral load, presumed route of infection and genetic makeup on immunological function. Whole blood, lymphocytes, and plasma/serum obtained from individuals with HAM/TSP, asymptomatic sero-positive individuals, sero-indeterminate individuals, as well as, healthy controls. We will assess virological and immunological parameters using these samples. In HTLV-1 seropositive individuals these studies will be extended to cerebrospinal fluid. 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. There is evidence that active HTLV-1 replication, through the retroviral life cycle with new virus integration, is occurring in vivo and contributes to the total HTLV-1 proviral load (PVL) in infected subjects. Recently it was shown that Raltegravir could inhibit cell-free and cell-to-cell transmission of HTLV-1 in vitro. Given the substantial clinical experience with its use in HIV-1 infection and particularly its excellent safety profile, we have initiated a phase I/II clinical trial to determine the effects of Raltegravir in vivo on HTLV-1 PVL and immune activation markers in patients with HAM/TSP. The primary outcome measure is HTLV-1 proviral load, was measured by quantitative PCR. Secondary outcome measures include safety and tolerability of Raltegravir, as assessed by clinical exam and standardized neurological disability scales as well as clinical laboratory studies. In addition, viral and immunologic outcome measures investigating the impact of Raltegravir on HTLV-1 biology and its effects on immune function have been preformed including HTLV-1 proviral load in different lymphocyte populations, the number of long terminal repeat (LTR) circles and HTLV-1 mRNA expression levels in freshly isolated PBMC, assays of spontaneous lymphoproliferation and T-cell phenotype analysis. As HAM/TSP is a chronic progressive inflammatory neurological disorder associated with virus-infected circulating T-cells that infiltrate the CNS, we initiated a proof of concept study to investigate if a T-cell receptor (TCR) repertoire signature in the peripheral blood PB could differentiate patients from HTLV-I seronegative healthy controls (HC). We therefore applied a recent unbiased molecular approach to investigate the TCR repertoire by HTS. We have validated this approach and have shown that the use of these molecular bar codes can lead to more accurate amplification and analysis of the T-cell clonal expansion profile without loss of real repertoire diversity. In addition, we are currently developing this technique for amplification of the TCR region using low numbers of cells (50.000) as we would obtain from compartments such as CSF. For HAM/TSP PBMC, over 225 million TCR sequences were generated on a HiSeq 2500 Illumina system and analyzed using molecular identifier groups-based error correction software (MiGEC). 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 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 HLA-A*0201 restricted 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. 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. Spinal cord atrophy measurements shows that the SSCA in HAM/TSP was thinner than healthy controls throughout the entire length of the cord. HTLV-I asymptomatic carriers were similar to that in HC. Moreover, we have shown the utility of this approach for quantitative spinal cord measurements in MS. Evaluation of 131 MS patients showed distinct patterns of atrophy in relapsing vs. progressive vs. HAM/TSP patients. RRMS patients were similar to control spinal cords while MS patients with progressive disease are intermediate between healthy volunteers and HAM/TSP patients. . Moreover, 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.