Teriflunomide and Epstein–Barr virus in a Spanish multiple sclerosis cohort: in vivo antiviral activity and clinical response

BackgroundEpstein–Barr virus (EBV) and human herpesvirus 6 (HHV-6) have been associated with multiple sclerosis (MS). Teriflunomide is an oral disease-modifying therapy approved for treatment of relapsing forms of MS. In the preclinical Theiler’s murine encephalitis virus model of MS, the drug demonstrated an increased rate of viral clearance versus the vehicle placebo. Furthermore, teriflunomide inhibits lytic EBV infection in vitro.Objective1. To evaluate the humoral response against EBV and HHV-6 prior to teriflunomide treatment and 6 months later. 2. To correlate the variation in the humoral response against EBV and HHV-6 with the clinical and radiological response after 24 months of treatment with teriflunomide. 3. To analyze the utility of different demographic, clinical, radiological, and environmental data to identify early biomarkers of response to teriflunomide.MethodsA total of 101 MS patients (62 women; mean age: 43.4 years) with one serum prior to teriflunomide onset and another serum sample 6 months later were recruited. A total of 80 had been treated for at least 24 months, 13 had stopped teriflunomide before 24 months, and 8 were currently under teriflunomide therapy but with less than 24 months of follow-up. We analyzed the levels of the viral antibodies titers abovementioned in serum samples with ELISA commercial kits, and the levels of serum neurofilament light chain (Nf-L).ResultsAntiviral antibody titers decreased for EBNA-1 IgG (74.3%), VCA IgG (69%), HHV-6 IgG (60.4%), and HHV-6 IgM (73.3%) after 6 months of teriflunomide. VCA IgG titers at baseline correlated with Nf-L levels measured at the same time (r = 0.221; p = 0.028) and 6 months later (r = 0.240; p = 0.017). We found that higher EBNA-1 titers (p = 0.001) and a higher age (p = 0.04) at baseline were associated with NEDA-3 conditions. Thus, 77.8% of patients with EBNA-1 >23.0 AU and >42.8 years (P50 values) were NEDA-3.ConclusionTreatment with teriflunomide was associated with a reduction of the levels of IgG antibody titers against EBV and HHV-6. Furthermore, higher EBNA-1 IgG titers prior to teriflunomide initiation were associated with a better clinical response

Herpesvirus Antibodies, Vitamin D and Short-Chain Fatty Acids: Their Correlation with Cell Subsets in Multiple Sclerosis Patients and Healthy Controls

Although the etiology of multiple sclerosis (MS) is still unknown, it is commonly accepted that environmental factors could contribute to the disease. The objective of this study was to analyze the humoral response to Epstein-Barr virus, human herpesvirus 6A/B and cytomegalovirus, and the levels of 25-hydroxyvitamin D (25(OH)D) and the three main short-chain fatty acids (SCFA), propionate (PA), butyrate (BA) and acetate (AA), in MS patients and healthy controls (HC) to understand how they could contribute to the pathogenesis of the disease. With this purpose, we analyzed the correlations among them and with different clinical variables and a wide panel of cell subsets. We found statistically significant differences for most of the environmental factors analyzed when we compared MS patients and HC, supporting their possible involvement in the disease. The strongest correlations with the clinical variables and the cell subsets analyzed were found for 25(OH)D and SCFAs levels. A correlation was also found between 25(OH)D and PA/AA ratio, and the interaction between these factors negatively correlated with interleukin 17 (IL-17)-producing CD4+ and CD8+ T cells in untreated MS patients. Therapies that simultaneously increase vitamin D levels and modify the proportion of SCFA could be evaluated in the future

Estudio del virus JC, agente causal de la leucoencafalopatía multifocal progresiva, en pacientes de esclerosis múltiple recurrente-remitente tratados con Natalizumab

La esclerosis múltiple (EM) es una enfermedad desmielinizante inflamatoria crónica que afecta al sistema nervioso central (SNC) y cuyo origen es presumiblemente autoinmune, siendo en los países desarrollados la segunda causa de discapacidad entre personas jóvenes después de los accidentes de tráfico. Durante los últimos años se han desarrollado, o actualmente están en ensayo clínico, una gran cantidad de fármacos para tratar esta patología. Entre todos ellos cabe destacar natalizumab, un anticuerpo monoclonal antagonista de la cadena α4 de la integrina dimérica α4/β1, presente en todos los leucocitos a excepción de los neutrófilos. Su mecanismo de acción consiste en impedir que estas células atraviesen la barrera hematoencefálica hacia el SNC, disminuyendo los procesos inflamatorios que están teniendo lugar en este órgano. Se caracteriza por su gran eficacia clínica comparada con el resto de fármacos del mercado; sin embargo, su administración ha sido asociada con la aparición de varios casos de leucoencefalopatía multifocal progresiva (LMP), una devastadora enfermedad desmielinizante del SNC causada por el virus JC. Hasta el 18 de abril de 2013 este fármaco ha sido administrado a más de 100.000 pacientes, entre los cuales se han diagnosticado unos 350 casos de LMP. Para determinar el riesgo de padecer esta enfermedad causada por el virus JC en los enfermos de EM tratados con natalizumab, la empresa farmacéutica que distribuye esta molécula ha elaborado un algoritmo en el que se tienen en cuenta 3 factores de riesgo: presencia/ausencia de anticuerpos anti-JC detectados por doble-ELISA, tiempo de exposición al tratamiento con natalizumab y uso previo de inmunosupresores. De tal forma que, aquellos pacientes que sean positivos a dichos anticuerpos anti-JC, hayan sido previamente tratados con inmunosupresores y hayan estado expuestos al fármaco durante más de 2 años, tendría un mayor riesgo de desarrollar la enfermedad [1 LMP:94 pacientes de EM tratados con natalizumab (IC: 1:130, 1:70)] frente al resto de grupos. El diagnóstico de la LMP se lleva a cabo mediante imágenes de resonancia magnética (RM), presencia de síntomas clínicos y detección del virus JC mediante reacción en cadena de la polimerasa (PCR) en líquido cefalorraquídeo (LCR), que suele estar presente en concentraciones muy bajas. El virus JC es un poliomavirus, no posee envuelta y su cápside icosaédrica alberga en su interior un pequeño genoma de ADN circular de doble hélice. Este genoma está organizado en 3 partes: una región codificante temprana, una tardía y una región reguladora (RR) común situada entre las dos anteriores. Esta RR contiene el origen de replicación (ORI) y los sitios de unión de distintos factores de transcripción que controlan la expresión génica del virus. Además, esta región determina la variante del virus y el tropismo celular del mismo. De esta forma en este trabajo distinguiremos entre: 1) la variante arquetípica: excretada por la orina de una gran parte de la población sana, caracterizada por ser no patogénica y cuya secuencia se divide en 7 fragmentos: ORI-A-B-C-D-E-F, 2) las variantes neurotrópicas, relacionadas con el desarrollo de LMP y compuesta por los mismos fragmentos que la arquetípica pero reordenados, según un patrón de deleciones y duplicaciones aún por determinar (ej: ORI-A-C-E-A-CE- F1-F2)..

Epstein-Barr Virus Load Correlates with Multiple Sclerosis-Associated Retrovirus Envelope Expression

pHERV-W ENV and syncytin-1, the envelope proteins of the human endogenous retrovirus W family (HERV-W), have been proposed as etiological factors for MS development. In addition, herpesviruses, such as the Epstein-Barr virus (EBV) and the human herpesvirus 6A/B (HHV-6A/B), have been also strongly associated with the disease. This work aims to study the possible link between viral loads and antibody titers against EBV and HHV-6A/B and the pHERV-W ENV/syncytin-1 protein/gene expression. For this purpose, we conducted a 12-month longitudinal study involving 98 RRMS patients. Peripheral blood samples were obtained from each patient. Serum antibody titers against EBV and HHV-6A/B were determined by ELISA, while viral loads were analyzed using qPCR. HLA MS-related alleles were also genotyped. pHERV-W ENV/syncytin-1 protein and gene expression levels in immune cells were assessed by flow cytometry and qPCR, respectively. We found that the 12-month variation of the pHERV-W ENV gene expression levels positively correlated with the variation of the EBV viral load, especially in those patients with high baseline EBV loads. Therefore, these results could support previous studies pointing to the transactivation of pHERV-W ENV by EBV. However, further studies are needed to better understand this possible relationship

Epidemiology of multiple sclerosis and vitamin D levels in Lanzarote, Canary Islands, Spain

Background Low levels of 25-hydroxyvitamin D (25(OH)D) have been described as one of the possible environmental factors involved in multiple sclerosis (MS) etiopathogenesis. Objectives To study epidemiology of MS and 25(OH)D serum levels of patients in Lanzarote (29°02′06″N), a region with high ultraviolet radiation values during the whole year which is located far apart from Iberian Peninsula (36°–43°N), but without genetic/ethnic differences with it. Methods Incidence in Lanzarote was assessed according to McDonald 2005 criteria between January 2008 and December 2015 and prevalence date was 12/31/15. For 25(OH)D serum levels analyses, samples from 60 MS patients and 60 healthy donors (HD) were collected monthly in a one-year prospective study. Results The prevalence of MS in Lanzarote was 50.0/100,000 and the incidence per year was 2.5/100,000. Median 25(OH)D levels values were 29.1 ng/ml for MS patients (maximum = 36.1 ng/ml, minimum = 22.5 ng/ml) and 27.1 ng/ml for HD (maximum = 34.8 ng/ml, minimum = 22.8 ng/ml). There were no significant differences between 25(OH)D serum levels between MS patients and HD. Conclusions Lanzarote possesses lower prevalence and incidence values than peninsular Spain. Moreover, 25(OH)D serum levels do not differ between MS patients and HD

Mitochondrial Impairments in Peripheral Blood Mononuclear Cells of Multiple Sclerosis Patients

Although impaired mitochondrial function has been proposed as a hallmark of multiple sclerosis (MS) disease, few studies focus on the mitochondria of immune cells. We aimed to compare the mitochondrial function of the peripheral blood mononuclear cells (PBMCs) from MS patients with (M+) and without (M−) lipid-specific oligoclonal immunoglobulin M bands (LS-OCMB), and healthydonors (HD). We conducted an exploratory cross-sectional study with 19 untreated MS patients (M+ = 9 and M− = 10) and 17 HDs. Mitochondrial superoxide anion production and mitochondrial mass in PBMCs were assessed without and with phytohemagglutinin by flow cytometry. The PBMCs’ mitochondrial function was analyzed using Seahorse technology. Superoxide anion production corrected by the mitochondrial mass was higher in MS patients compared with HDs (p = 0.011). Mitochondrial function from M+ patients showed some impairments compared with M− patients. Without stimulus, we observed higher proton leak (p = 0.041) but lower coupling efficiency (p = 0.041) in M+ patients; and under stimulation, lower metabolic potential ECAR (p = 0.011), and lower stressed OCR/ECAR in the same patients. Exclusively among M+ patients, we described a higher mitochondrial dysfunction in the oldest ones. The mitochondrial impairments found in the PBMCs from MS patients, specifically in M+ patients, could help to better understand the disease’s physiopathology

A Polymorphism Within the MBP Gene Is Associated With a Higher Relapse Number in Male Patients of Multiple Sclerosis

Myelin basic protein (MBP) is thought to be one of the key autoantigens in multiple sclerosis (MS) development. A recent study described the association of the single nucleotide polymorphism (SNP) rs12959006, within the MBP gene, with a higher risk of relapse and worse prognosis. We aim at studying potential associations of this SNP to MS in an independent population. Clinical data of the first 5 years of the disease were collected retrospectively from 291 MS confirmed patients. MBP polymorphism rs12959006 was genotyped in all patients. Associations with EDSS, number of relapses and serology for Herpesvirus 6 (HHV-6) and Epstein Barr (EBV) viruses were studied. Lymphocyte activation measured by CD69 expression was also analyzed according to sex and rs12959006 genotype. The rs12959006 polymorphism contributed significantly to a higher number of relapses at 5 years after onset only in male patients (rs12959006 ∗ TT β = 0.74 [0.36-1.09]; p = 7 × 10 -5). Titers of anti-HHV6 IgG antibodies showed also a mild association with relapses, both in male and female patients (β = 0.01 [0.01-0.02]; p = 3.7 × 10 -8). Both the genetic variation in MBP and HHV-6 infection aid in predicting a higher number of relapses during the first years of MS. The association described in MBP rs12959006 ∗ T is exclusive to male patient