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. Various inflammatory neurologic diseases are associated with viral infections. These agents may cause direct damage of infected cells associated with immunological alterations such as chronic activation, immunodeficiency and infiltration of inflammatory cells into the CNS that underlie the pathogenesis of inflammatory neurologic diseases. Therefore, characterization of local immune responses that are associated with the inflammatory milieu may also provide evidence or a pathogenic signature of an immunopathogenic process in virus-associated neurologic diseases. 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. Basic laboratory investigations have demonstrated the importance of the cytokine IL-15 in the life and death of lymphocytes and for its role in autoimmune disorders such as HAM/TSP. IL-15 is pivotally involved in the survival of CD8+ memory T-cells including self-directed cells and we have shown that in HAM/TSP, IL-15 is essential for the survival of HLA class I restricted virus antigen-specific effector and memory CD8+ T-cells. Collaborative research by the Viral Immunology Section, NINDS and the Metabolism Branch, NCI are completing a phase I clinical trial for the treatment of HAM/TSP using Hu MiK-beta-1 that blocks the action of IL-15. We have assessed the effects of intravenously administered Hu-MiK-beta-1 on the cellular immune response in patients with HAM/TSP, with particular focus on virus-specific memory CD8+ T cells. Secondary outcomes to be measured are clinical responses, including toxicity, and the effect on CD4+CD25+ T regulatory cells. As IL-15 over expression has been demonstrated in patients in a wide variety of autoimmune disorders including, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis and psoriasis, the successful application of Hu MiK-beta-1 in HAM/TSP has significant and broad therapeutic implications. 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, which will be measured by quantitative PCR. Secondary outcome measures include safety and tolerability of Raltegravir, which will be 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 will be measured 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 (P= 0.0039). Despite the biased T-cell profile in HAM/TSP patients, a shared identical CDR3 amino acid sequence signature was not found across patients. Of note, relatedness among clones was observed in HAM/TSP patients by phylogenetic tree analysis. Our TCR -chain sequencing analysis suggested that an individual immune T-cell repertoire signature exists in patients with this neuro-immune inflammatory disorder. 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.
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