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Epidemiological studies have demonstrated that Epstein-Barr virus (EBV) is a known etiologic risk factor, and perhaps prerequisite, for the development of MS. EBV establishes life-long latent infection in a subpopulation of memory B cells. Although the role of memory B cells in the pathobiology of MS is well established, studies characterizing EBV-associated mechanisms of B cell inflammation and disease pathogenesis in EBV (+) B cells from MS patients are limited. Accordingly, we analyzed spontaneous lymphoblastoid cell lines (SLCLs) from multiple sclerosis patients and healthy controls to study host-virus interactions in B cells, in the context of an individual’s endogenous EBV. We identify differences in EBV gene expression and regulation of both viral and cellular genes in SLCLs. Our data suggest that EBV latency is dysregulated in MS SLCLs with increased lytic gene expression observed in MS patient B cells, especially those generated from samples obtained during “active” disease. Moreover, we show increased inflammatory gene expression and cytokine production in MS patient SLCLs and demonstrate that tenofovir alafenamide, an antiviral that targets EBV replication, decreases EBV viral loads, EBV lytic gene expression, and EBV-mediated inflammation in both SLCLs and in a mixed lymphocyte assay. Collectively, these data suggest that dysregulation of EBV latency in MS drives a pro-inflammatory, pathogenic phenotype in memory B cells and that this response can be attenuated by suppressing EBV lytic activation. This study provides further support for the development of antiviral agents that target EBV-infection for use in MS.

Background: Dramatic improvements in visualization of cortical (especially subpial) multiple sclerosis (MS) lesions allow assessment of impact on clinical course. Objective: Characterize cortical lesions by 7 tesla (T) T2*-/T1-weighted magnetic resonance imaging (MRI); determine relationship with other MS pathology and contribution to disability. Methods: Sixty-four adults with MS (45 relapsing–remitting/19 progressive) underwent 3 T brain/spine MRI, 7 T brain MRI, and clinical testing. Results: Cortical lesions were found in 94% (progressive: median 56/range 2–203; relapsing–remitting: 15/0–168; p = 0.004). Lesion distribution across 50 cortical regions was nonuniform ( p = 0.006), with highest lesion burden in supplementary motor cortex and highest prevalence in superior frontal gyrus. Leukocortical and white matter lesion volumes were strongly correlated ( r = 0.58, p < 0.0001), while subpial and white matter lesion volumes were moderately correlated ( r = 0.30, p = 0.002). Leukocortical ( p = 0.02) but not subpial lesions ( p = 0.40) were correlated with paramagnetic rim lesions; both were correlated with spinal cord lesions ( p = 0.01). Cortical lesion volumes (total and subtypes) were correlated with expanded disability status scale, 25-foot timed walk, nine-hole peg test, and symbol digit modality test scores. Conclusion: Cortical lesions are highly prevalent and are associated with disability and progressive disease. Subpial lesion burden is not strongly correlated with white matter lesions, suggesting differences in inflammation and repair mechanisms.

Summary Background Progressive multifocal leukoencephalopathy, a rare disease of the CNS caused by JC virus and occurring in immunosuppressed people, is typically fatal unless adaptive immunity is restored. JC virus is a member of the human polyomavirus family and is closely related to the BK virus. We hypothesised that use of partly HLA-matched donor-derived BK virus-specific T cells for immunotherapy in progressive multifocal leukoencephalopathy would be feasible and safe. Methods We did an open-label, single-cohort pilot study in patients (aged 18 years or older) with clinically definite progressive multifocal leukoencephalopathy and disease progression in the previous month at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Overlapping peptide libraries derived from large T antigen and major capsid protein VP1 of BK virus with high sequence homology to JC virus counterparts were used to generate polyomavirus-specific T cells cross-recognising JC virus antigens. Polyomavirus-specific T cells were manufactured from peripheral blood mononuclear cells of first-degree relative donors aged 18 years or older. These cells were administered to patients by intravenous infusion at 1 × 106 polyomavirus-specific T cells per kg, followed by up to two additional infusions at 2 × 106 polyomavirus-specific T cells per kg. The primary endpoints were feasibility (no manufacturing failure based on meeting release criteria, achieving adequate numbers of cell product for clinical use, and showing measurable antiviral activity) and safety in all patients. The safety monitoring period was 28 days after each infusion. Patients were followed up with serial MRI for up to 12 months after the final infusion. This trial is registered at ClinicalTrials.gov , NCT02694783 . Findings Between April 7, 2016, and Oct 19, 2018, 26 patients were screened, of whom 12 were confirmed eligible and received treatment derived from 14 matched donors. All administered polyomavirus-specific T cells met the release criteria and recognised cognate antigens in vitro. 12 patients received at least one infusion, ten received at least two, and seven received a total of three infusions. The median on-study follow-up was 109·5 days (range 23–699). All infusions were tolerated well, and no serious treatment-related adverse events were observed. Seven patients survived progressive multifocal leukoencephalopathy for longer than 1 year after the first infusion, whereas five died of progressive multifocal leukoencephalopathy within 3 months. Interpretation We showed that generation of polyomavirus-specific T cells from healthy related donors is feasible, and these cells can be safely used as an infusion for adoptive immunotherapy of progressive multifocal leukoencephalopathy. Although not powered to assess efficacy, our data provide additional support for this strategy as a potential life-saving therapy for some patients. Funding Intramural Research Program of the National Institute of Neurological Disorders and Stroke of the NIH.

Highlights • Spinal cord atrophy is prevalent across multiple sclerosis phenotypes. • It correlates with disability, especially in relapsing-remitting patients. • This correlation can be demonstrated both cross-sectionally and longitudinally. • Cervical atrophy is highly associated with disability and disease progression. • Thoracic atrophy contributes to improved correlation and radiological subgrouping., Objective We sought to characterize spinal cord atrophy along the entire spinal cord in the major multiple sclerosis (MS) phenotypes, and evaluate its correlation with clinical disability. Methods Axial T1-weighted images were automatically reformatted at each point along the cord. Spinal cord cross‐sectional area (SCCSA) were calculated from C1-T10 vertebral body levels and profile plots were compared across phenotypes. Average values from C2-3, C4-5, and T4-9 regions were compared across phenotypes and correlated with clinical scores, and then categorized as atrophic/normal based on z-scores derived from controls, to compare clinical scores between subgroups. In a subset of relapsing-remitting cases with longitudinal scans these regions were compared to change in clinical scores. Results The cross-sectional study consisted of 149 adults diagnosed with relapsing-remitting MS (RRMS), 49 with secondary-progressive MS (SPMS), 58 with primary-progressive MS (PPMS) and 48 controls. The longitudinal study included 78 RRMS cases. Compared to controls, all MS groups had smaller average regions except RRMS in T4-9 region. In all MS groups, SCCSA from all regions, particularly the cervical cord, correlated with most clinical measures. In the RRMS cohort, 22% of cases had at least one atrophic region, whereas in progressive MS the rate was almost 70%. Longitudinal analysis showed correlation between clinical disability and cervical cord thinning. Conclusions Spinal cord atrophy was prevalent across MS phenotypes, with regional measures from the RRMS cohort and the progressive cohort, including SPMS and PPMS, being correlated with disability. Longitudinal changes in the spinal cord were documented in RRMS cases, making it a potential marker for disease progression. While cervical SCCSA correlated with most disability and progression measures, inclusion of thoracic measurements improved this correlation and allowed for better subgrouping of spinal cord phenotypes. Cord atrophy is an important and easily obtainable imaging marker of clinical and sub-clinical progression in all MS phenotypes, and such measures can play a key role in patient selection for clinical trials.

Background: Progressive Multifocal Encephalopathy (PML) is a subacute infection of the central nervous system (CNS) mediated by John Cunningham (JC) polyomavirus (PyV). The disease is frequently fatal, unless adaptive immunity to JC virus is restored. The disease typically occurs in immunosuppressed patients (e.g. HIV/AIDS). In the modern era, patients with hematological malignancies treated with rituximab and/or fludarabine or following immunosuppressive therapy for transplant or autoimmunity (e.g. multiple sclerosis (MS) or Crohn's disease) are also at risk. JC-PML can also occur in patients with genetic defects of immunity. Specific treatments do not exist and immunocompromised patients with history of cancer or genetic defects of immunity have no realistic chance for rapid recovery of their ability to fight infections. In subjects with Rituximab-related PML the case-fatality ratio is >90%. Survivors can be left with severe neurological disabilities. Methods: Based on our previously established procedure for generation of multi-virus virus-specific T cells using overlapping peptide libraries (pepmixes) as immunogens, we have developed dedicated polyomavirus-specific T cells (PyVST) targeting polyomavirus BK large T (LT) and Viral Protein 1 (VP1) antigens and highly cross-reactive with the structurally-homologous JC LT and VP1 proteins (see Figure). We hypothesized that adoptive transfer of donor-derived PyVSTs could be safely used for therapy of patients with refractory JC-PML. We have developed a pilot study to test the feasibility and safety of adoptive immunotherapy strategy using PyVSTs from the partially matched healthy 1st degree relative donors (sibling, parent or offspring; NIH study 16-N-0072). Adults diagnosed with PML who have no other treatment options were eligible. Patients with MS or HIV were excluded. PyVSTs were generated from donor blood leukocytes cultured for 14 days in G-rex flasks upon stimulation with BK LT and VP1 pepmixes. Subjects underwent baseline physical examination, MRI and lumbar puncture. Upon enrollment subjects received an intravenous infusion of a fixed dose of 1x10e6 (+/-10%) PyVST cells/kg. Safety monitoring period was 28 days after each infusion. Subjects were eligible for up to two additional infusions (dose 2x10e6 PyVSTs/kg) a minimum of 28 days apart if no toxicities were observed. Serial MRI and lumbar punctures were performed to monitor response. Subjects were followed for 12 months after the last infusion. Results: Nine subjects have been enrolled and treated with at least one infusion of PyVSTs under this protocol and the trial is still accruing subjects. No immediate infusion reactions or adverse events have been observed within the safety monitoring period. No CNS immune system reconstitution syndrome (IRIS) has occurred. One subject received a single infusion and withdrew from the study following Day 14 visit due to unwillingness to travel. One subject received two doses of PyVSTs with stabilization of the disease, not requiring the 3rd infusion. This subject was withdrawn from the study just prior to the 1 year follow up visit, as he was diagnosed with the stage IV lung cancer and entered hospice. Seven subjects received 3 infusions of PyVSTs. 3 of these 7 patients died of refractory PML, all >30 days after the 3rd dose of T cells. At the time of this report one subject completed the study at one year with stable PML. The remaining subjects are in the follow-up phase of the protocol. Conclusions: We report that partially matched T cells targeting PyVs generated from healthy related donors can be safely used for adoptive immunotherapy of severely immunocompromised patients with PML. Multiple infusions of T cells are very well tolerated at the doses of up to 2x10e6 PyVST cells/kg, displaying excellent safety profile without adverse events. Furthermore, our data suggest possible efficacy of this strategy as a life-saving therapy for patients who otherwise face a dismal prognosis. Disclosures No relevant conflicts of interest to declare.