Abstract Body

Background: Progressive multifocal leukoencephalopathy (PML), caused by JC virus (JCV), is an opportunistic infection in immunocompromised hosts and its pathogenesis remains elusive. Recently, PML affects multiple sclerosis (MS) patients treated with natalizumab, a monoclonal antibody against alpha-4 integrins which prevents entry of inflammatory cells in the central nervous system. We sought to uncover the site and prevalence of JCV reactivation and determine JCV-specific cellular response during prolonged exposure to the drug. Methodology: We enrolled 39 JCV-seropositive MS patients, including 32 on natalizumab monotherapy for 18-20 months (n=14), 22-25 months (n=7) and >36 months (n=11), 2 on interferon-beta monotherapy >36 months and 5 untreated MS patients as controls. We performed QPCR in CSF, blood and urine for JCV DNA and we determined JCV specific T-cell responses using enzyme-linked immunospot (ELISpot) and intracellular cytokine staining (ICS) essays, ex vivo and after in vitro stimulation with JCV peptides Results: JCV DNA was detected in the CSF of 3/35 (8.6%) subjects tested (1 in 18-20 and 2 in >36 months on natalizumab), who had no symptoms or MRI lesions consistent with PML. JCV DNA was detected in peripheral blood mononuclear cells (PBMC) of 10/39 (25.6%) subjects but not in plasma. JCV DNA load was higher in CD34+ cells compared to B Cells (p=0.0064), T cells (p=0.0035) and polymorphonuclear (PMN) cells (p=0.0043) and in monocytes compared to T cells (p=0.0048) and PMN (p=0.008). Viruria was detected in 8/39 (20.5%) patients. JCV-specific CD4+ T-cells were detected ex vivo more frequently in subjects with JCV DNA in PBMC (p=0.027). JCV-specific T cells were detected more frequently by ICS than ELISpot ex vivo [26/39 (66.7%) vs 6/39 (15.3%);p<0.001] and after in vitro stimulation [39/39 (100%) vs 33/38 tested (86.8%);p=0.03]. Both assays were significantly more frequently positive after in vitro stimulation than ex vivo (p<0.001). JCV-specific CD4+ T-cells were more frequently detected than CD8+ T-cells after in vitro stimulation [39/39(100%) vs 34/39(87.2%); p=0.05]. Conclusions: Asymptomatic JCV reactivation may occur in CSF of MS patients on prolonged natalizumab treatment. JCV is associated with multiple leukocyte subpopulations and viral load may be higher in CD34+ cells and monocytes compared to other cells. JCV detection in plasma or urine may not be adequate markers for viral reactivation. Presence of the virus in the bloodstream may trigger a CD4+ mediated immune response. JCV-specific cellular response is highly prevalent in MS patients and its detection may be enhanced after in vitro stimulation with JCV peptides.