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

Background Epstein–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. Objective 1. 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. Methods A 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). Results Antiviral 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. Conclusion Treatment 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.


Introduction
Multiple sclerosis (MS) is a disease of unknown origin.Classically, it has been considered the result of the interaction of one or several environmental factors, which would act in the early stages of life on people predisposed from the genetic point of view.Many viruses have been associated with MS over the years.However, viruses of the Herpesviridae family, specifically Epstein-Barr virus (EBV), have accumulated the most evidence in recent years.The higher seroprevalence of EBV in patients with MS compared with healthy subjects has been widely described (1).In addition, the risk would increase in subjects who contract EBV in adulthood and develop infectious mononucleosis (IM) compared with those who are infected in childhood and do not develop IM (2).It has been hypothesized that dysregulation of EBV-infected B cells could induce leptomeningeal inflammation, which could contribute to subpial lesions and gray matter pathology in MS (3,4).Thus, it has been observed that high levels of antibodies against EBV would be associated with an increase in the activity of lesions in MRI (5, 6), and greater development of brain atrophy, particularly of the cortical gray matter (6,7).There are currently not many options to inhibit EBV replication, but previous in vitro studies have shown that teriflunomide could exhibit inhibitory activity on EBV (8).
Teriflunomide is an oral immunomodulator approved for the treatment of relapsing-remitting MS (RRMS) and clinically isolated syndrome (CIS).The efficacy and safety of teriflunomide in patients with RRMS were established in phase 2 (NCT01487096) and phase 3 (TEMSO-NCT00134563, TOWER-NCT00751881, and TENERE-NCT00883337) clinical trials (9)(10)(11)(12) and in patients with CIS in the TOPIC study (NCT00622700) (13,14).Recently, the findings from the phase 3 TERIS study (NCT03122652) showed that treatment significantly reduced the time to first clinical event in patients with radiologically isolated syndrome (RIS) (15).Teriflunomide reversibly inhibits the enzyme dihydroorotate dehydrogenase (DHODH), a key mitochondrial enzyme in the de novo synthesis of pyrimidines (16).It has been shown to have anti-inflammatory but also antiviral properties against different viruses: EBV (8), cytomegalovirus (17), herpes simplex virus 1 and 2 (18,19), footand-mouth disease virus (20), and against poliovirus BK (21).It has been proposed that this antiviral effect would be achieved by inhibiting viral replication.Recent studies that describe antiviral compounds that act at the level of the de novo pyrimidine biosynthetic pathway, such as teriflunomide, raise the hypothesis that the mode of action of these molecules may be based on the inhibition of DHODH (22).
Thus, the objectives of this study were (1) to evaluate the in vivo antiviral activity of teriflunomide in MS patients through the evaluation of the variation of IgG antibody titers against EBNA-1 and VCA of EBV and IgG and IgM against HHV-6 between the baseline visit (before treatment initiation) and after 6 months of treatment with teriflunomide (2), to associate the variation of these titers with the clinical and radiological response after 24 months of treatment with teriflunomide, and (3) to search for early response biomarkers to teriflunomide.

Design
A multicenter longitudinal study was performed.We recruited MS patients under the following inclusion criteria: a diagnostic of RRMS according to the McDonald criteria (23), with serum samples collected prior to the initiation of teriflunomide and 6 months later.
Serum samples were collected in dry tubes; then, they were aliquoted and aliquots were frozen at −80°C.

Patients
They were recruited from the MS Units of the following hospitals: Hospital Clıńico San Carlos, Hospital Universitario Ramón y Cajal, Hospital Universitario de Getafe, Hospital General Universitario Gregorio Marañoń, Hospital Universitario de Torrejón, Hospital A ́lvaro Cunqueiro, and Hospital Universitario Vall d'Hebron, all of them in Spain.

Ethics statement
This study was approved by the local Ethic Committee of the Hospital Clıńico San Carlos (ComitéE ́tico de Investigacioń Clıńica del Hospital Clıńico San Carlos).All the patients recruited received and signed written informed consent.All experiments were performed in accordance with relevant guidelines and regulations.

Response criteria
Clinical response (absence of disability progression and relapses), NEDA-3 (no evidence of disease activity: patients without relapses, disability progression, and new T2 lesions or Gd+ lesions), and therapeutic failure (disability progression and/ or ≥2 relapses) were evaluated after 2 years of follow-up.Relapses: a worsening of neurological impairment or an appearance of a new symptom or abnormality attributable to MS; they lasted at least 24 h and were preceded by a stability period of at least 1 month.Progression: according to EDSS score prior to teriflunomide initiation (1): EDSS = 0: ≥1.5 points (2); EDSS ≥1 and ≤5: ≥1 point (3); EDSS ≥5.5: ≥0.5 points.Magnetic resonance imaging (MRI) examination was performed within 1 month prior to teriflunomide initiation according to the protocols established in each center and 12 and 24 months later.

Viral antibody quantification
Serum samples were analyzed in an automated ELISA processing system (DS2, Dynex Technologies, USA) using the following commercial tests: anti-EBNA-1 and anti-VCA IgG (Trinity Biotech, USA) and anti-HHV-6A/B IgG and IgM (Vidia, Ltd., Czech Republic).Each sample was analyzed in duplicate for each test.Results were expressed in artificial units (AU), as we have previously described (24, 25).

Statistical analysis
Differences in categorical variables were analyzed with the chisquare or two-tailed Fisher's exact test.Differences in continuous variables were assessed with the Mann-Whitney U-test.A multiple logistic regression model was performed to eliminate the effect of the putative confounding variables in the univariate tests.p values below 0.05 were considered significant.Statistical analyses were performed with SPSS for Windows (Ver.15.0) software (SPSS Inc.) and with GraphPad Prism 6.0 software (GraphPad Prism Inc., San Diego, CA).

Patients recruited according to the inclusion criteria
As we can see in Figure 1, we recruited 101 MS patients.A total of 13 stopped teriflunomide before 24 months, 10 of which were due to lack of efficacy: four due to activity, three due to progression, and three due to activity and progression; all of them were considered non-clinical responders.The other three patients abandoned teriflunomide due to a lack of tolerance, and they were not included in the clinical response analysis.Other eight patients were under teriflunomide treatment, but they did not reach 24 months of follow-up yet.Thus, we performed the clinical response analysis with 90 patients.We also had radiological data (number of T2 lesions and gadolinium-enhanced lesions at 0, 12, and 24 months) from 82/90 patients to establish NEDA-3 conditions.Basal patient data are in Table 1.

Variation of antiviral IgG titers and Nf-L levels after 6 months with teriflunomide
Antiviral antibody prevalences and titers in samples collected prior to teriflunomide initiation are in Table 2.After 6 months of teriflunomide treatment, antiviral antibody titers decreased in 61/ 101 (60.4%)MS patients for HHV-6 IgG, 75/101 (74.3%) for HHV-6 IgM, 74/101 (73.3%) for EBNA-1 IgG and 69/100 (69%) for VCA IgG (Figure 2).No statistically significant differences were found between both genders or between those that were above or below 43.4 years (median value) for any of the viruses.
We did not find any statistical significant difference between the levels of Nf-L prior to the initiation of teriflunomide treatment and after 6 months of therapy (11.0 ± 6.7 pg/ml vs. 11.6 ± 6.0; p = 0.499) (Figure 3).
When we analyzed the correlation between the titers of each one of the viral antibodies prior to the initiation of teriflunomide treatment with the Nf-L levels for the basal sample and for the 6-month sample (Figure 4), we found a statistical significant correlation for VCA IgG in both cases: r = 0.221 (p = 0.028) for the basal sample and r = 0.240 (p = 0.017) for the 6-month sample.

Clinical and radiological response after 2 years of treatment with teriflunomide
A percentage of 68.9% (62/90) were clinical responders (no progression, no relapses), and 14.4% (13/90) had therapeutic failure (≥2 relapses and/or progression) after 2 years of teriflunomide treatment.A percentage of 45.1% (37/82) were NEDA-3 (no progression, no relapses, no new T2 lesions, no gadoliniumenhanced lesions) after 2 years of teriflunomide treatment.We did not find any statistical significant association between the variation of the titers of any of the viruses between the samples collected at 0M and 6M with the clinical response or the NEDA-3 condition after 2 years of treatment.Furthermore, the increase or decrease of the Nf-L levels was not also associated with the response to teriflunomide.

Demographic, clinical, radiological, and environmental data to identify early biomarkers of response to teriflunomide
When we analyzed the variables that could predict NEDA-3 conditions at treatment initiation, we found the following statistical associations in the univariate analysis (p < 0.05): starting age, disease duration, EBNA-1 titers, and number of gadoliniumenhanced lesions, all of them at the recruitment (before starting teriflunomide treatment).Therefore, those MS patients that were older, with long-lasting disease, higher EBNA-1 titers, and lower number of gadolinium-enhanced lesions were better responders.When we performed the multivariate analysis, only two variables     EBNA-1 IgG, VCA IgG, HHV-6 IgG, and HHV-6 IgM titers.Variation of the titers in each patient between samples collected prior to teriflunomide treatment (0M) and after 6 months of treatment (6M).

Discussion
In the last years, some authors have indicated that part of the benefits observed in MS patients treated with teriflunomide could be due to its antiviral properties.Gilli et al. evaluated the effect of teriflunomide in the Theiler model.The authors analyzed the effects of treatment on CNS viral load, intrathecal immune response, and disability progression.They observed that teriflunomide had both anti-inflammatory and antiviral properties, although they did not observe any impact on intrathecal antibody synthesis.Teriflunomide demonstrated a higher percentage of viral clearance compared with placebo, indicating that this treatment could have a prophylactic and therapeutic role against CNS viral infections (27).In our study, teriflunomide reduced the levels of IgG antibody titers against EBNA-1 and VCA of EBV and IgG and IgM against HHV-6 after 6 months of treatment in a real-life cohort of Spanish MS patients.These results are relevant since EBV has been proposed as the leading cause of MS (1).Our group recently published a paper in which we described that neither interferonbeta, glatiramer acetate, nor natalizumab had a significant effect on EBNA-1 and VCA IgG titers after 2 years of follow-up (28).In this previous study, we found that EBNA-1 IgG titers decreased in the 56.5% of patients treated with natalizumab, 39.1% of patients treated with interferon-beta, and 36.2% of patients treated with glatiramer acetate, after 6 months of treatment, which is significantly lower than the 73.3% found in the current cohort of MS patients treated with teriflunomide.The in vivo results of this study could be explained, at least in part, by the results of previous in vitro studies.In a paper published by Bilger et al. (8), teriflunomide inhibited cellular proliferation and promoted apoptosis, in EBV-transformed B cells in vitro at a clinically relevant dose.In addition, teriflunomide prevented the development of EBV-induced lymphomas in both a humanized mouse model and a xenograft model.Furthermore, teriflunomide inhibited lytic EBV infection in vitro both by preventing the initial steps of lytic viral reactivation and by blocking lytic viral DNA replication.Our results also support a previous in vivo study performed in a small cohort of MS patients.Zivadinov et al. (29) designed a longitudinal study with 30 MS who were starting treatment with teriflunomide.The authors found that MS patients experienced a significant decrease in IgG antibody titers against EBNA-1 (p = 0.003) and VCA (p = 0.05) after 12 months of treatment.MS patients who showed a greater decrease in IgG antibody titers against VCA and EBNA-1 from the baseline sample developed less loss of cortical volume (p < 0.001 and p = 0.02, respectively) and gray matter (p = 0.004 for both antibodies).
These antiviral properties could undoubtedly be of great interest in MS since, apart from the possible role of EBV in the etiopathogenesis of the disease, viral CNS infections are associated with other disease-modifying therapies (DMTs).It is the case of progressive multifocal leukoencephalopathy (PML) in which the causative agent is the JC virus (30-32).Given that teriflunomide can cross the blood-brain barrier (BBB) with 1-2% of serum concentrations (in the range of 2.5-4.1 mM) reaching the CNS (33), the usefulness of teriflunomide, at least in patients at risk of developing PML, could be hypothesized.This question has been addressed recently in a clinical trial (ClinicalTrials.govIdentifier: NCT01970410).In that study, authors assessed the safety and efficacy of rapid transition, from natalizumab to teriflunomide in MS patients.A total of 55 MS patients were enrolled, and 51% of them completed 24 months of teriflunomide.There were no cases of PML.The authors concluded that the washout-free transition of natalizumab to teriflunomide was an efficacious and safe strategy for patients at risk of developing PML (34).
In this study, we also evaluated the levels of Nf-L at basal visit and after 6 months of teriflunomide treatment.As we described above, we did not find any significant change in Nf-L levels.Since in the work of Bjornevik et al. (1) the authors assessed the possible temporal relation between EBV infection and Nf-L increase, we decided to analyze in our study the possible correlations between these two variables in the first 6 months with teriflunomide.In the previous study, it is described that NfL levels in EBV-negative MS patients at baseline were similar to those of controls before and around the time of EBV infection but increased after EBV infection.In our study, we found a correlation between VCA IgG titers at baseline and the Nf-L levels measured at the same time and 6 months later, but not with EBNA-1.This result is concordant with a previous study performed by Uher et al. (35); in this paper, authors found an association between VCA IgG levels and quartiles with sNfL levels, but not with EBNA-1.Thus, although more studies are needed to afford this question, EBV seems to be involved in those mechanisms that are associated with the release of Nf-L into the CSF and serum.
Finally, we analyzed the utility of different demographic, clinical, radiological, and environmental data to identify early biomarkers of response to teriflunomide.In the current MS scenario, with an increasing number of DMTs, it is crucial to identify the most appropriate treatment for each patient to ensure a good therapeutic response and to reduce costs.As we described previously, only EBNA-1 titers at baseline and the starting age of the treatment remain significant after the multivariate analysis.Those MS patients with both variables above the 50th percentile had a higher probability of reaching NEDA-3 conditions, since more than 75% of them were free of progression, relapses, new T2 lesions, and gadolinium-enhanced lesions, after 2 years of teriflunomide treatment.The results of the early biomarkers of response to teriflunomide highlight the possible antiviral role of this treatment since better results were obtained in MS patients with higher EBNA-1 IgG titers.
In conclusion, treatment with teriflunomide was associated with a reduction of the levels of IgG antibody titers against EBNA-1 and VCA of EBV and IgG and IgM against HHV-6 after 6 months of treatment in a real-life cohort of Spanish MS patients.A correlation between VCA IgG titers at baseline and the Nf-L levels measured at the same time and 6 months later was found.Furthermore, our results describe two variables that could be used as early predictors of NEDA-3 condition in MS patients that are going to initiate teriflunomide therapy.

FIGURE 1
FIGURE 1Study design.MS patients included in each one of the analysis.

TABLE 1
Basal characteristics of the patients recruited at the onset of teriflunomide treatment.

TABLE 2
Serologies of the virus included in the study prior to the initiation of teriflunomide treatment.
*There was a missing value.