Multiple Sclerosis and Related Disorders (2014) 3, 372–374
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/msard
Multiple sclerosis therapy and Epstein–Barr virus antibody titres Joel Raffela,n, Ruth Dobsona, Arie Gafsona, Miriam Mattosciob, Paolo Murarob, Gavin Giovannonia a
Queen Mary University of London, Blizard Institute, Barts and the London School of Medicine and Dentistry, UK b Department of Medicine, Division of Brain Sciences, Centre for Neuroscience, Wolfson Neuroscience Laboratories, Imperial College, London, UK Received 29 April 2013; received in revised form 11 June 2013; accepted 13 December 2013
KEYWORDS Biological markers; EBV-encoded nuclear antigen 1; Herpesvirus 4, Human; Interferon-beta; Multiple sclerosis; Natalizumab
1.
Abstract Background: Anti-Epstein–Barr virus (EBV) nuclear antigen-1 (anti-EBNA-1) IgG antibody titres have been found to correlate with MRI and clinical measures of disease activity in MS. Despite being a putative biomarker of disease activity, the effect of disease modifying drugs on antiEBNA-1 IgG titre has not yet been determined. Methods: In this study, we investigated the effect of interferon-beta and natalizumab therapy on prospective sera anti-EBNA-1 IgG titres, using a quantitative ELISA, in patients with relapsing-remitting MS. Results: For both the interferon-beta and natalizumab group, there was no significant difference between pre-therapy and post-therapy anti-EBNA-1 IgG titre. There was also no significant difference between the groups with regard to mean percentage change in anti-EBNA1 IgG titre over 12 months of treatment. Conclusions: This study suggests that anti-EBNA-1 IgG titre is unlikely to be a good surrogate marker for disease activity in patients on disease modifying drugs. & 2013 Elsevier B.V. All rights reserved.
Introduction
There is need for accessible and effective biomarkers of disease activity in multiple sclerosis (MS). There is also
Abbreviations: EBV, Epstein–Barr virus; anti-EBNA-1, anti-Epstein– Barr virus (EBV) nuclear antigen-1 n Correspondence to: Centre for Neuroscience and Trauma, Blizard Institute of Cell and Molecular Science, 4 Newark Street, London E1 2AT, UK. Tel.: +44 7814632510; fax: +44 20 7882 2200. E-mail address: [email protected] (J. Raffel). 2211-0348/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msard.2013.12.004
increasing evidence that both infection with, and the ongoing immune response to, Epstein–Barr virus (EBV) contributes to the pathogenesis of MS (Salvetti et al., 2009). Several studies have therefore evaluated serological markers of EBV activity as putative biomarkers of MS disease activity (Castellazzi et al., 2010; Farrell et al., 2009; Höllsberg et al., 2005; Ingram et al., 2010; Lindsey et al., 2009; Wandinger et al., 2000). A cross-sectional study found a positive correlation between anti-Epstein–Barr virus nuclear antigen-1 (antiEBNA-1) IgG antibody titres and MRI measures of disease activity (Farrell et al., 2009). This study also demonstrated
Multiple sclerosis therapy and Epstein–Barr virus antibody titres a positive correlation between anti-EBNA-1 IgG titre and Expanded Disability Status Scale score (EDSS), although this was not reproduced in a subsequent study (Ingram et al., 2010). However, despite being a putative biomarker of disease activity, the effect of disease modifying drugs (DMDs) on anti-EBNA-1 IgG titre has not yet been well characterised. In this prospective study, we measured anti-EBNA-1 IgG titres in patient with relapsing-remitting MS (RRMS), both before and 12 months after the initiation of treatment with interferon-beta (IFN-β) or natalizumab. Since both IFN-β and natalizumab have been shown to decrease relapse rate and MRI activity in MS, we hypothesised that these DMDs would also decrease anti-EBNA-1 IgG titres. In addition, we wanted to explore the CNS compartmentalisation theory of EBV infection in MS, as proposed by Serafini et al. (2007). If the CNS is indeed a site of EBV reactivation in MS, and one then prevents trafficking of lymphocytes into the brain with natalizumab, one may expect EBV reactivation and proliferation in the brain without a corresponding CNS EBV-specific cytotoxic CD8 + response. This would potentially result in release of EBV antigens from the CNS into the periphery, acting as an immune booster and causing an increase in peripheral antiEBNA-1 titres, much like that observed with natalizumab and CNS JCV infection (Trampe et al., 2012). In contrast this should not occur with interferon beta, since it does not prevent CNS lymphocyte migration to the same extent as natalizumab.
2.
373 prospective individual anti-EBNA-1 IgG titre was analysed using repeated measures ANOVA.
3.
Results
There was no difference between the natalizumab and IFN-β groups, when comparing mean baseline anti-EBNA-1 IgG titre (natalizumab vs. IFN-β: mean7SD=14,78675647 vs. 15,8627 6819 AU/mL; p=0.71), mean 12-month post-treatment antiEBNA-1 IgG titre (natalizumab vs. IFN-β: mean7SD=13,6247 5977 vs. 16,71077756 AU/mL; p=0.33), or mean percentage change in anti-EBNA-1 IgG titre (natalizumab vs. IFN-β: mean percentage change7SD= 8.7%711.5% vs. +3.4%719.3%; p=0.10). IFN-β had no effect on individual anti-EBNA-1 IgG titre, when comparing baseline titre with those 12 months posttreatment (p= 0.80; see Fig. 1). Similarly, natalizumab had no effect on individual anti-EBNA-1 IgG titre, when comparing baseline titres with those 6 and 12 months posttreatment (p =0.462 at 6 months; p= 0.118 at 12 months; see Fig. 2). Of the patients receiving natalizumab, only patients 1 and 8 had any relapses post-treatment initiation. Patient 1 had three relapses in year one, and patient 8 had one relapse. There was no significant difference in the percentage change in EBNA1 IgG titre between these two patients and the rest of the natalizumab cohort.
Materials and methods
Blood samples were collected from 10 treatment-naïve patients prior to initiation of IFN-β (Betaferon, Avonex, or Rebif), and 10 patients due to initiate natalizumab, after informed consent. Patients starting natalizumab were either treatment naïve or had not received any treatment for MS for at least 6 months. The baseline sample was drawn immediately prior to treatment initiation, whilst the subsequent sample was drawn at 12 months follow-up. Six patients on natalizumab had two follow-up samples taken at both 6 and 12 months. Serum was divided into aliquots and stored at 80 1C. Repeated free-thaw cycles were avoided. This study had ethical approval from either the South East Multi-Centre Research Ethics Committee (reference 04/01/033) or the Queen Charlotte0 s & Chelsea Research Ethics Committee (reference 09/H0707/18). Anti-EBNA-1 IgG titres were measured using a commercially available quantitative ELISA kit (DiaSorin; Salugia, Italy). All assays were performed in duplicate by one operator (JR), who was blinded to the treatment allocation of the samples. The mean pre and post-treatment anti-EBNA-1 IgG titre7standard deviation was calculated for the natalizumab and the IFN-β groups. All data were tested for normality prior to statistical analyses (Shapiro–Wilks normality test). Data which were not normally distributed were log transformed and retested to confirm normality. An unpaired t test was used to compare titres between treatment groups. The effect of IFN-β on prospective individual anti-EBNA-1 IgG titre was analysed using a paired t test. The effect of natalizumab on
Fig. 1.
Effect of IFN-β on prospective anti-EBNA-1 IgG titre.
Fig. 2. titre.
Effect of natalizumab on prospective anti-EBNA-1 IgG
374
4.
J. Raffel et al.
Discussion
This study found no effect of DMDs on anti-EBNA-1 IgG titre. Anti-EBNA-1 IgG titres have been previously shown to correlate with MRI measures of MS disease activity in a cross-sectional study (Farrell et al., 2009). Given that both IFN-β and natalizumab decrease both relapse rate and MRI activity, it is surprising that anti-EBNA-1 IgG titres do not decrease post-treatment. Therefore, it seems unlikely that anti-EBNA-1 IgG would be a useful biomarker of MS disease activity in those on DMDs. Further conclusions can be drawn from these data. Firstly, it has previously been suggested that the mechanism of action of IFN-β is antiviral (Dhib-Jalbut, 2002); these data argue against an antiviral effect of IFN-β on EBV of sufficient magnitude and duration to affect serum IgG anti-EBNA1 antibody titres. Secondly, other groups have argued for the CNS compartmentalisation theory of EBV infection in MS (Serafini et al., 2007). If this were true, then one should expect anti-EBNA-1 IgG titres to increase after starting natalizumab, much in the same way that anti-JCV antibody titres increase in those on natalizumab with dysregulated JCV CNS infection (Trampe et al., 2012). Since anti-EBNA-1 IgG titres show—if anything—a downward trend post-natalizumab, this argues strongly against dysregulated EBV infection in the CNS. The main limitation of this study is that the sample size is underpowered to detect modest effects. However, the study results argue against an effect of DMDs on anti-EBNA-1 IgG.
Conflicts of interest PAM has received speaker0 s honoraria frp, Biogen Idec. MM has received travel support from Biogen Idec. GG has received research grant support from Bayer-Schering Healthcare, BiogenIdec, GW Pharma, Merck Serono, Merz, Novartis, Teva and Sanofi-Aventis. GG has also received personal compensation for participating on Advisory Boards in relation to clinical trial design, trial steering committees and data and safety monitoring committees from Bayer-Schering Healthcare, Biogen-Idec, Eisai, Elan, Fiveprime, Genzyme, Genentech, GSK, GW Pharma, Ironwood, Merck-Serono, Novartis, Pfizer, Roche, Sanofi-Aventis, Synthon BV, Teva, UCB Pharma and Vertex Pharmaceuticals. The other authors have no conflicts of interest.
Acknowledgements This work was supported by a generous grant from the Roan Charitable Trust, The natalizumab patients sample
collection was part of a project funded by the Italian MS Society (FISM; Ref. No. Project Grant 2010/R/24, Ref. No. Training Fellowship 2010/B/10); RD is funded by an Association of British Neurologists/MS Society of Great Britain Clinical Research Fellowship.
References Castellazzi M, Tamborino C, Cani A, Negri E, Baldi E, Seraceni S, et al. Epstein–Barr virus-specific antibody response in cerebrospinal fluid and serum of patients with multiple sclerosis. Mult Scler 2010;16(7):883–7. Dhib-Jalbut S. Mechanisms of action of interferons and glatiramer acetate in multiple sclerosis. Neurology 2002;58:S3–9. (review). Farrell RA, Antony D, Wall GR, Clark DA, Fisniku L, Swanton J, et al. Humoral immune response to EBV in multiple sclerosis is associated with disease activity on MRI. Neurology 2009;73 (1):32–8. Höllsberg P, Kusk M, Bech E, Hansen HJ, Jakobsen J, Haahr S. Presence of Epstein–Barr virus and human herpesvirus 6B DNA in multiple sclerosis patients: associations with disease activity. Acta Neurol Scand 2005;112(6):395–402. Ingram G, Bugert JJ, Loveless S, Robertson NP. Anti-EBNA-1 IgG is not a reliable marker of multiple sclerosis clinical disease activity. Eur J Neurol 2010;17(11):1386–9. Lindsey JW, Hatfield LM, Crawford MP, Patel S. Quantitative PCR for Epstein–Barr virus DNA and RNA in multiple sclerosis. Mult Scler 2009;15(2):153–8. Salvetti M, Giovannoni G, Aloisi F. Epstein–Barr virus and multiple sclerosis. Curr Opin Neurol 2009;22(3):201–6. (review). Serafini B, Rosicarelli B, Franciotta D, Magliozzi R, Reynolds R, Cinque P, et al. Dysregulated Epstein–Barr virus infection in the multiple sclerosis brain. J Exp Med 2007;204(12):2899–912. Trampe AK, Hemmelmann C, Stroet A, Haghikia A, Hellwig K, Wiendl H, et al. Anti-JC virus antibodies in a large German natalizumabtreated multiple sclerosis cohort. Neurology 2012;78 (22):1736–42. Wandinger K, Jabs W, Siekhaus A, Bubel S, Trillenberg P, Wagner H, et al. Association between clinical disease activity and Epstein– Barr virus reactivation in MS. Neurology 2000;55(2):178–84.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/msard
Multiple sclerosis therapy and Epstein–Barr virus antibody titres Joel Raffela,n, Ruth Dobsona, Arie Gafsona, Miriam Mattosciob, Paolo Murarob, Gavin Giovannonia a
Queen Mary University of London, Blizard Institute, Barts and the London School of Medicine and Dentistry, UK b Department of Medicine, Division of Brain Sciences, Centre for Neuroscience, Wolfson Neuroscience Laboratories, Imperial College, London, UK Received 29 April 2013; received in revised form 11 June 2013; accepted 13 December 2013
KEYWORDS Biological markers; EBV-encoded nuclear antigen 1; Herpesvirus 4, Human; Interferon-beta; Multiple sclerosis; Natalizumab
1.
Abstract Background: Anti-Epstein–Barr virus (EBV) nuclear antigen-1 (anti-EBNA-1) IgG antibody titres have been found to correlate with MRI and clinical measures of disease activity in MS. Despite being a putative biomarker of disease activity, the effect of disease modifying drugs on antiEBNA-1 IgG titre has not yet been determined. Methods: In this study, we investigated the effect of interferon-beta and natalizumab therapy on prospective sera anti-EBNA-1 IgG titres, using a quantitative ELISA, in patients with relapsing-remitting MS. Results: For both the interferon-beta and natalizumab group, there was no significant difference between pre-therapy and post-therapy anti-EBNA-1 IgG titre. There was also no significant difference between the groups with regard to mean percentage change in anti-EBNA1 IgG titre over 12 months of treatment. Conclusions: This study suggests that anti-EBNA-1 IgG titre is unlikely to be a good surrogate marker for disease activity in patients on disease modifying drugs. & 2013 Elsevier B.V. All rights reserved.
Introduction
There is need for accessible and effective biomarkers of disease activity in multiple sclerosis (MS). There is also
Abbreviations: EBV, Epstein–Barr virus; anti-EBNA-1, anti-Epstein– Barr virus (EBV) nuclear antigen-1 n Correspondence to: Centre for Neuroscience and Trauma, Blizard Institute of Cell and Molecular Science, 4 Newark Street, London E1 2AT, UK. Tel.: +44 7814632510; fax: +44 20 7882 2200. E-mail address: [email protected] (J. Raffel). 2211-0348/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msard.2013.12.004
increasing evidence that both infection with, and the ongoing immune response to, Epstein–Barr virus (EBV) contributes to the pathogenesis of MS (Salvetti et al., 2009). Several studies have therefore evaluated serological markers of EBV activity as putative biomarkers of MS disease activity (Castellazzi et al., 2010; Farrell et al., 2009; Höllsberg et al., 2005; Ingram et al., 2010; Lindsey et al., 2009; Wandinger et al., 2000). A cross-sectional study found a positive correlation between anti-Epstein–Barr virus nuclear antigen-1 (antiEBNA-1) IgG antibody titres and MRI measures of disease activity (Farrell et al., 2009). This study also demonstrated
Multiple sclerosis therapy and Epstein–Barr virus antibody titres a positive correlation between anti-EBNA-1 IgG titre and Expanded Disability Status Scale score (EDSS), although this was not reproduced in a subsequent study (Ingram et al., 2010). However, despite being a putative biomarker of disease activity, the effect of disease modifying drugs (DMDs) on anti-EBNA-1 IgG titre has not yet been well characterised. In this prospective study, we measured anti-EBNA-1 IgG titres in patient with relapsing-remitting MS (RRMS), both before and 12 months after the initiation of treatment with interferon-beta (IFN-β) or natalizumab. Since both IFN-β and natalizumab have been shown to decrease relapse rate and MRI activity in MS, we hypothesised that these DMDs would also decrease anti-EBNA-1 IgG titres. In addition, we wanted to explore the CNS compartmentalisation theory of EBV infection in MS, as proposed by Serafini et al. (2007). If the CNS is indeed a site of EBV reactivation in MS, and one then prevents trafficking of lymphocytes into the brain with natalizumab, one may expect EBV reactivation and proliferation in the brain without a corresponding CNS EBV-specific cytotoxic CD8 + response. This would potentially result in release of EBV antigens from the CNS into the periphery, acting as an immune booster and causing an increase in peripheral antiEBNA-1 titres, much like that observed with natalizumab and CNS JCV infection (Trampe et al., 2012). In contrast this should not occur with interferon beta, since it does not prevent CNS lymphocyte migration to the same extent as natalizumab.
2.
373 prospective individual anti-EBNA-1 IgG titre was analysed using repeated measures ANOVA.
3.
Results
There was no difference between the natalizumab and IFN-β groups, when comparing mean baseline anti-EBNA-1 IgG titre (natalizumab vs. IFN-β: mean7SD=14,78675647 vs. 15,8627 6819 AU/mL; p=0.71), mean 12-month post-treatment antiEBNA-1 IgG titre (natalizumab vs. IFN-β: mean7SD=13,6247 5977 vs. 16,71077756 AU/mL; p=0.33), or mean percentage change in anti-EBNA-1 IgG titre (natalizumab vs. IFN-β: mean percentage change7SD= 8.7%711.5% vs. +3.4%719.3%; p=0.10). IFN-β had no effect on individual anti-EBNA-1 IgG titre, when comparing baseline titre with those 12 months posttreatment (p= 0.80; see Fig. 1). Similarly, natalizumab had no effect on individual anti-EBNA-1 IgG titre, when comparing baseline titres with those 6 and 12 months posttreatment (p =0.462 at 6 months; p= 0.118 at 12 months; see Fig. 2). Of the patients receiving natalizumab, only patients 1 and 8 had any relapses post-treatment initiation. Patient 1 had three relapses in year one, and patient 8 had one relapse. There was no significant difference in the percentage change in EBNA1 IgG titre between these two patients and the rest of the natalizumab cohort.
Materials and methods
Blood samples were collected from 10 treatment-naïve patients prior to initiation of IFN-β (Betaferon, Avonex, or Rebif), and 10 patients due to initiate natalizumab, after informed consent. Patients starting natalizumab were either treatment naïve or had not received any treatment for MS for at least 6 months. The baseline sample was drawn immediately prior to treatment initiation, whilst the subsequent sample was drawn at 12 months follow-up. Six patients on natalizumab had two follow-up samples taken at both 6 and 12 months. Serum was divided into aliquots and stored at 80 1C. Repeated free-thaw cycles were avoided. This study had ethical approval from either the South East Multi-Centre Research Ethics Committee (reference 04/01/033) or the Queen Charlotte0 s & Chelsea Research Ethics Committee (reference 09/H0707/18). Anti-EBNA-1 IgG titres were measured using a commercially available quantitative ELISA kit (DiaSorin; Salugia, Italy). All assays were performed in duplicate by one operator (JR), who was blinded to the treatment allocation of the samples. The mean pre and post-treatment anti-EBNA-1 IgG titre7standard deviation was calculated for the natalizumab and the IFN-β groups. All data were tested for normality prior to statistical analyses (Shapiro–Wilks normality test). Data which were not normally distributed were log transformed and retested to confirm normality. An unpaired t test was used to compare titres between treatment groups. The effect of IFN-β on prospective individual anti-EBNA-1 IgG titre was analysed using a paired t test. The effect of natalizumab on
Fig. 1.
Effect of IFN-β on prospective anti-EBNA-1 IgG titre.
Fig. 2. titre.
Effect of natalizumab on prospective anti-EBNA-1 IgG
374
4.
J. Raffel et al.
Discussion
This study found no effect of DMDs on anti-EBNA-1 IgG titre. Anti-EBNA-1 IgG titres have been previously shown to correlate with MRI measures of MS disease activity in a cross-sectional study (Farrell et al., 2009). Given that both IFN-β and natalizumab decrease both relapse rate and MRI activity, it is surprising that anti-EBNA-1 IgG titres do not decrease post-treatment. Therefore, it seems unlikely that anti-EBNA-1 IgG would be a useful biomarker of MS disease activity in those on DMDs. Further conclusions can be drawn from these data. Firstly, it has previously been suggested that the mechanism of action of IFN-β is antiviral (Dhib-Jalbut, 2002); these data argue against an antiviral effect of IFN-β on EBV of sufficient magnitude and duration to affect serum IgG anti-EBNA1 antibody titres. Secondly, other groups have argued for the CNS compartmentalisation theory of EBV infection in MS (Serafini et al., 2007). If this were true, then one should expect anti-EBNA-1 IgG titres to increase after starting natalizumab, much in the same way that anti-JCV antibody titres increase in those on natalizumab with dysregulated JCV CNS infection (Trampe et al., 2012). Since anti-EBNA-1 IgG titres show—if anything—a downward trend post-natalizumab, this argues strongly against dysregulated EBV infection in the CNS. The main limitation of this study is that the sample size is underpowered to detect modest effects. However, the study results argue against an effect of DMDs on anti-EBNA-1 IgG.
Conflicts of interest PAM has received speaker0 s honoraria frp, Biogen Idec. MM has received travel support from Biogen Idec. GG has received research grant support from Bayer-Schering Healthcare, BiogenIdec, GW Pharma, Merck Serono, Merz, Novartis, Teva and Sanofi-Aventis. GG has also received personal compensation for participating on Advisory Boards in relation to clinical trial design, trial steering committees and data and safety monitoring committees from Bayer-Schering Healthcare, Biogen-Idec, Eisai, Elan, Fiveprime, Genzyme, Genentech, GSK, GW Pharma, Ironwood, Merck-Serono, Novartis, Pfizer, Roche, Sanofi-Aventis, Synthon BV, Teva, UCB Pharma and Vertex Pharmaceuticals. The other authors have no conflicts of interest.
Acknowledgements This work was supported by a generous grant from the Roan Charitable Trust, The natalizumab patients sample
collection was part of a project funded by the Italian MS Society (FISM; Ref. No. Project Grant 2010/R/24, Ref. No. Training Fellowship 2010/B/10); RD is funded by an Association of British Neurologists/MS Society of Great Britain Clinical Research Fellowship.
References Castellazzi M, Tamborino C, Cani A, Negri E, Baldi E, Seraceni S, et al. Epstein–Barr virus-specific antibody response in cerebrospinal fluid and serum of patients with multiple sclerosis. Mult Scler 2010;16(7):883–7. Dhib-Jalbut S. Mechanisms of action of interferons and glatiramer acetate in multiple sclerosis. Neurology 2002;58:S3–9. (review). Farrell RA, Antony D, Wall GR, Clark DA, Fisniku L, Swanton J, et al. Humoral immune response to EBV in multiple sclerosis is associated with disease activity on MRI. Neurology 2009;73 (1):32–8. Höllsberg P, Kusk M, Bech E, Hansen HJ, Jakobsen J, Haahr S. Presence of Epstein–Barr virus and human herpesvirus 6B DNA in multiple sclerosis patients: associations with disease activity. Acta Neurol Scand 2005;112(6):395–402. Ingram G, Bugert JJ, Loveless S, Robertson NP. Anti-EBNA-1 IgG is not a reliable marker of multiple sclerosis clinical disease activity. Eur J Neurol 2010;17(11):1386–9. Lindsey JW, Hatfield LM, Crawford MP, Patel S. Quantitative PCR for Epstein–Barr virus DNA and RNA in multiple sclerosis. Mult Scler 2009;15(2):153–8. Salvetti M, Giovannoni G, Aloisi F. Epstein–Barr virus and multiple sclerosis. Curr Opin Neurol 2009;22(3):201–6. (review). Serafini B, Rosicarelli B, Franciotta D, Magliozzi R, Reynolds R, Cinque P, et al. Dysregulated Epstein–Barr virus infection in the multiple sclerosis brain. J Exp Med 2007;204(12):2899–912. Trampe AK, Hemmelmann C, Stroet A, Haghikia A, Hellwig K, Wiendl H, et al. Anti-JC virus antibodies in a large German natalizumabtreated multiple sclerosis cohort. Neurology 2012;78 (22):1736–42. Wandinger K, Jabs W, Siekhaus A, Bubel S, Trillenberg P, Wagner H, et al. Association between clinical disease activity and Epstein– Barr virus reactivation in MS. Neurology 2000;55(2):178–84.
