During the last two decades, an association between progressive multifocal leukoencephalopathy (PML) and diseases modifying therapies (DMT) for multiple sclerosis (MS) has come into light. PML is a demyelinating disease of the central nervous system (CNS) associated with JC virus (JCV) infection in the oligodendrocytes. Several cases of PML associated with natalizumab, a monoclonal antibody (anti-CD49d), have been reported. An increased PML risk in MS patients treated with other DMT was reported. Recently, a new monoclonal antibody was licensed for MS treatment: ocrelizumab (anti-CD20) and some cases of PML during this treatment were reported. Monoclonal antibodies such as natalizumab and ocrelizumab are likely to generate immunological impairments leading to JCV reactivation and PML development. During MS, matrix metalloproteinase (MMP)-9 plays a role in the breakdown of the blood-brain barrier (BBB), thus facilitating the entry of inflammatory cells into the central nervous system (CNS). A reduction in the levels of molecules involved in BBB disruption, such as MMP-9, could interfere with CD8+ T-lymphocyte immune surveillance mechanisms in the CNS, predisposing MS patients to JCV reactivation and PML development. MMP-9 plasma activity and mRNA expression in peripheral blood mononuclear cells (PBMC), JCV-DNA and T-lymphocyte phenotypic modifications will be evaluated in multiple sclerosis (MS) patients starting or already under natalizumab or ocrelizumab treatment. MMP-9 activity will be assessed by zymography in plasma samples while MMP-9 mRNA expression will be evaluated through quantitative real time PCR (qPCR) in PBMC. JCV-DNA will be detected through qPCR in urine and plasma samples and PBMC. B and T-lymphocyte phenotype will be assessed by flow cytometry. These findings could contribute to understand PML pathogenesis under DMT, defining the role of MMP-9 in this setting.
The serological, immunological and virological monitoring of MS patients under DMT seems to be helpful in order to understand the pathogenesis of PML and the specific deficits of the host-immune system, leading to JCV reactivation. Furthermore, searching for early markers of JCV reactivation and host immunological responses represents an essential issue to address in order to improve the management of MS treatment and increase the safety profile of DMT. In this study, host-pathogen interaction will be longitudinally assessed in order to monitor immunological modifications induced by natalizumab or ocrelizumab treatments and their relations to JCV reactivation. This approach will improve the clinical management of MS patients and increase the safety profile of monoclonal antibody treatments. To date, PML risk assessment and monitoring during natalizumab treatment is based on clinical data, MRI scanning and JCV-specific antibody detection. Indeed, the routine monitoring of JCV serostatus using the Stratify JCV test is now standard of care for MS patients (Iannetta et al., 2016b). However, due to high JCV seroprevalence both in the general population (60%) and MS patients (51%), the rare occurrence of PML (3.72 per 1000 natalizumab MS patients) makes this tool insufficient in defining the disease development risk (Bozic et al., 2011; Serana et al., 2014) underlining the need for appropriate biomarkers to better predict PML development. In previous papers, we evaluated the relationship between JCV reactivation and the alterations of T-lymphocyte subsets in a cohort of MS patients under natalizumab treatment (Iannetta et al., 2016b; Zingaropoli et al., 2018).
JCV serology alone is not sufficient for the diagnosis of JCV infection and should be associated with JCV-DNA detection in body fluids (such as blood or urine) in order to truly identify JCV infected patients (Berger et al., 2013; Major et al., 2013). We observed a modification of CD8 subset distribution, with accumulation of CD8 central memory, effector memory, and effector subsets in peripheral blood. Furthermore, CD8 effectors tended to increase in natalizumab treated MS patients with JCV-DNA detection in plasma or urine.
This study will contribute to clarify the immune pathogenesis of PML caused by JCV reactivation by comparing immunological changes induced by natalizumab and ocrelizumab. This approach will be also intended to identify early markers of JCV reactivation and PML onset, which will be helpful for clinical management of MS patients under DMT, improving safety profile.
This study will also give a deeper insight in the role of MMP-9 as a predictive tool for PML onset, during monoclonal antibody treatment. Natalizumab and ocrelizumab effect on MMP-9 activity, mRNA expression and intracellular secretion by B and T lymphocyte will be evaluated. All these parameters will be correlated to JCV reactivation in blood and urine sample.