Viral Immune signatures from cerebrospinal fluid extracellular vesicles and particles in HAM and other chronic neurological diseases

Background and objectivesExtracellular vesicles and particles (EVPs) are released from virtually all cell types, and may package many inflammatory factors and, in the case of infection, viral components. As such, EVPs can play not only a direct role in the development and progression of disease but can also be used as biomarkers. Here, we characterized immune signatures of EVPs from the cerebrospinal fluid (CSF) of individuals with HTLV-1-associated myelopathy (HAM), other chronic neurologic diseases, and healthy volunteers (HVs) to determine potential indicators of viral involvement and mechanisms of disease.MethodsWe analyzed the EVPs from the CSF of HVs, individuals with HAM, HTLV-1-infected asymptomatic carriers (ACs), and from patients with a variety of chronic neurologic diseases of both known viral and non-viral etiologies to investigate the surface repertoires of CSF EVPs during disease.ResultsSignificant increases in CD8+ and CD2+ EVPs were found in HAM patient CSF samples compared to other clinical groups (p = 0.0002 and p = 0.0003 compared to HVs, respectively, and p = 0.001 and p = 0.0228 compared to MS, respectively), consistent with the immunopathologically-mediated disease associated with CD8+ T-cells in the central nervous system (CNS) of HAM patients. Furthermore, CD8+ (p < 0.0001), CD2+ (p < 0.0001), CD44+ (p = 0.0176), and CD40+ (p = 0.0413) EVP signals were significantly increased in the CSF from individuals with viral infections compared to those without.DiscussionThese data suggest that CD8+ and CD2+ CSF EVPs may be important as: 1) potential biomarkers and indicators of disease pathways for viral-mediated neurological diseases, particularly HAM, and 2) as possible meditators of the disease process in infected individuals

Comparison of qPCR with ddPCR for the Quantification of JC Polyomavirus in CSF from Patients with Progressive Multifocal Leukoencephalopathy

Background: Lytic infection of oligodendrocytes by the human JC polyomavirus (JCPyV) results in the demyelinating disease called progressive multifocal leukoencephalopathy (PML). The detection of viral DNA in the cerebrospinal fluid (CSF) by PCR is an important diagnostic tool and, in conjunction with defined radiological and clinical features, can provide diagnosis of definite PML, avoiding the need for brain biopsy. The main aim of this study is to compare the droplet digital PCR (ddPCR) assay with the gold standard quantitative PCR (qPCR) for the quantification of JC viral loads in clinical samples. Methods: A total of 62 CSF samples from 31 patients with PML were analyzed to compare the qPCR gold standard technique with ddPCR to detect conserved viral DNA sequences in the JCPyV genome. As part of the validation process, ddPCR results were compared to qPCR data obtained in 42 different laboratories around the world. In addition, the characterization of a novel triplex ddPCR to detect viral DNA sequence from both prototype and archetype variants and a cellular housekeeping reference gene is described. Triplex ddPCR was used to analyze the serum from six PML patients and from three additional cohorts, including 20 healthy controls (HC), 20 patients with multiple sclerosis (MS) who had never been treated with natalizumab (no-NTZ-treated), and 14 patients with MS who were being treated with natalizumab (NTZ-treated); three from this last group seroconverted during the course of treatment with natalizumab. Results: JCPyV DNA was detected only by ddPCR for 5 of the 62 CSF samples (8%), while remaining undetected by qPCR. For nine CSF samples (15%), JCPyV DNA was at the lower limit of quantification for qPCR, set at <250 copies/mL, and therefore no relative quantitation could be determined. By contrast, exact copies of JCPyV for each of these samples were quantified by ddPCR. No differences were observed between qPCR and ddPCR when five standardized plasma samples were analyzed for JCPyV in 42 laboratories in the United States and Europe. JCPyV-DNA was undetected in all the sera from HC and MS cohorts tested by triplex ddPCR, while serum samples from six patients with PML tested positive for JCPyV. Conclusion: This study shows strong correlation between ddPCR and qPCR with increased sensitivity of the ddPCR assay. Further work will be needed to determine whether multiplex ddPCR can be useful to determine PML risk in natalizumab-treated MS patients

Immunophenotypic characterization of CSF B cells in virus-associated neuroinflammatory diseases

Intrathecal antibody synthesis is a well-documented phenomenon in infectious neurological diseases as well as in demyelinating diseases, but little is known about the role of B cells in the central nervous systems. We examined B cell and T cell immunophenotypes in CSF of patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) compared to healthy normal donors and subjects with the other chronic virus infection and/or neuroinflammatory diseases including HIV infection, multiple sclerosis (MS) and progressive multifocal leukoencephalopathy. Antibody secreting B cells (ASCs) were elevated in HAM/TSP patients, which was significantly correlated with intrathecal HTLV-1-specific antibody responses. High frequency of ASCs was also detected in patients with relapsing-remitting multiple sclerosis (RRMS). While RRMS patients showed significant correlations between ASCs and memory follicular helper CD4+ T cells, CD4+CD25+ T cells were elevated in HAM/TSP patients, which were significantly correlated with ASCs and HTLV-1 proviral load. These results highlight the importance of the B cell compartment and the associated inflammatory milieu in HAM/TSP patients where virus-specific antibody production may be required to control viral persistence and/or may be associated with disease development

Detection of CD4⁺CD25⁺ T cells in CSF of chronic virus infection and/or neuroinflammatory diseases.

<p>(A) Comparison of frequency of CD4⁺CD25⁺ T cells in CSF using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean. (B) Comparison of frequency of CD4⁺CD25⁺ T cells in peripheral blood using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean. (C) Comparison of frequency of FoxP3 in peripheral blood CD4⁺CD25⁺ T cells using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean. (D) Comparison of frequency of CTLA-4 in peripheral blood CD4⁺CD25⁺ T cells using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean.</p

Correlation between ASC and CD4⁺ T cell subsets.

<p>(A) Correlation of ASCs with memory Tfh cells in CSF of patients with MS, HAM/TSP, HIV and PML using Spearman’s rank correlation test. (B) Correlation of ASCs with CD4⁺CD25⁺ T cells in CSF of patients with HAM/TSP and PML using Spearman’s rank correlation test. (C) Comparison of frequency of CD4⁺CD25⁺ T cells and ASCs in CSF of HAM/TSP patients between non-progressors and progressors using Mann-Whitney Test. The horizontal line represents the mean. (D) Correlation of ASCs in CSF B cells with disease duration in HAM/TSP patients (n = 32) using Spearman’s rank correlation test.</p

Antibody responses against HTLV-1 in CSF of HTLV-1-infected subjects.

<p>(A) Comparison of antibody responses against HTLV-1 Gag, Env and Tax in serum and CSF of ACs and HAM/TSP patients using Mann-Whitney Test. (B) Comparison of CSF/serum anti-HTLV-1 Gag, Env and Tax antibody ratio of HAM/TSP patients and ACs using Paired T test or Mann-Whitney Test. All the data were obtained from HAM/TSP patients (n = 44) and ACs (n = 4). The horizontal line represents the mean. (C) Correlation of ASCs in CSF B cells with anti-Gag, anti-Env and anti-Tax antibody index in HAM/TSP patients (n = 11) using Spearman’s rank correlation test.</p

Detection of B cells in CSF of chronic virus infection and/or neuroinflammatory diseases.

<p>(A) Comparison of frequencies of B cells (left) and B cell/monocyte ratio (right) in CSF using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean. (B) Detection of ASCs subset in B cells of CSF. (Left) Representative dot plots of IgD and CD27 staining in CSF CD19⁺ B cells of a ND and a HAM/TSP patient. IgD^- CD27⁺⁺ subsets (red rectangles) represent ASCs. (C) Comparison of frequencies of ASCs (left) and absolute number of ASCs (right) in CSF using Kruskal-Wallis test with Dunn’s test. The horizontal line represents the mean.</p

Detection of antibody secreting B cells in CSF.

<p>Detection of antibody secreting B cells in CSF.</p