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Natalizumab modulates the humoral response against HERV-Wenv73–88 in a follow-up study of Multiple Sclerosis patients
Journal of the Neurological Sciences, In Press, Corrected Proof, Available online 8 July 2015
Multiple Sclerosis (MS) is a heterogeneous disorder of the central nervous system (CNS) that begins as an inflammatory autoimmune disorder mediated by auto-reactive lymphocyte followed by microglial activation and chronic degeneration. The etiology of Multiple Sclerosis (MS) is unknown but several data support the hypothesis of possible infectious agents which may act as a trigger for the pathogenic cascade. Human endogenous retrovirus (HERV-W/MSRV), Epstein Barr Virus (EBV) and Mycobacterium avium ss. paratuberculosis (MAP) have been associated to Multiple Sclerosis. In this study, we evaluated the humoral response against different peptides: the human endogenous retrovirus HERV-Wenv73–88, MAP106c121–132 from MAP, EBNA1 400–413 from EBV and the homologous human peptide MBP85–98 in a cohort of MS patients treated with natalizumab. Results showed a statistically significant difference in the response against the HERV-W peptide in MS patients after two years of natalizumab treatment.
- Ab response against EBNA1, MAP106c, MBP and HERV-Wenv was evaluated in MS patients.
- No difference was observed during natalizumab treatment for EBNA1, MAP106c, and MBP.
- Difference in the Ab response was observed for HERV-Wenv during natalizumab exposure.
- Natalizumab treatment reduces Ab production against HERV-Wenv in MS patients.
Keywords: Multiple Sclerosis, Natalizumab therapy, HERV-Wenv, Epstein Barr Virus (EBV), Mycobacterium avium ss. paratuberculosis (MAP), Antibodies, ELISA.
The etiology of Multiple Sclerosis (MS) is unknown. Different infectious agents may trigger the pathogenic cascade. HERV-W/MSRV, Epstein Barr Virus (EBV) and Mycobacterium avium ss. paratuberculosis (MAP) are the most intriguing intracellular pathogens studied in MS patients. EBV has been proposed as a causal factor in MS  , longitudinal studies showed that increased titer of anti-EBV antibodies occur long before MS onset. MAP is responsible of Johne's disease in ruminants  and it has been associated with several autoimmune diseases as Crohn, type I diabetes and Hashimoto Thyroiditis  and MS in Sardinian patients  . HERV-W/MSRV has been reported to be associated with MS development and prognosis through encoding a powerful immunopathogenic envelope protein (env) that activates a pro-inflammatory and immune-mediated cascade causing MS  . Natalizumab is a humanized monoclonal antibody (mAb) indicated as monotherapy in relapse remitting MS (RRMS) patients. It acts by blocking the binding between the α4 subunit of the α4β1 of the circulating lymphocytes very late antigen-4 (VLA-4) and α4β7 integrins with their ligand, the vascular cell adhesion molecule 1 (VCAM-1)  . In this study we evaluated the humoral response against different peptides: HERV-Wenv73–88, MAP106 c 121–132, EBNA1400–413 and the human homologous peptide MBP85–98 in a longitudinal cohort study of MS patients treated with natalizumab and in a cohort of post-natalizumab patients.
2. Materials and methods
MS Sardinian patients diagnosed according to international criteria  were enrolled at the MS Centre for Diagnosis and Treatment, Dept. of Clinical and Experimental Medicine (Neurology), University Hospital of Sassari, Italy. Peripheral venous blood was collected before natalizumab treatment (T0) and after three (T3), six (T6), twelve (T12) and 24 (T24) months of therapy. Initially the cohort included 48 MS patients at T0 (F/M = 3.4; mean age 41.98 ± 10.5), 48 at T3, 47 at T6, 41 at T12 and only 12 at T24 ( Table 1 A). Another group included 27 MS patients (F/M = 3.5; mean age 40 ± 10.8) who had finished 24-month natalizumab therapy at least 3 months prior to collection ( Table 1 B). 41 sex matched healthy blood donors (BD) at the Blood Transfusion Center of Sassari were used as control subjects for MAP, EBV, and HERV-Wenv blood circulation in the overall population (F/M = 3.55, mean age 41 ± 10.47).
|A||n||Mean||SD||Range (min–max)||B||n||Mean||SD||Range (min–max)|
|Sex (females/males)||37/11||Sex (females/males)||21/6|
|Years of disease||12.26||8.35||2–32||Years of disease||10.82||3.37||6–18|
|Disease form (RRMS)||RR||Disease form (RRMS)||RR|
The local ethic committee approved the study (Prot. N. 2.150/2015, Azienda Sanitaria Locale 1, Sassari, Italy) and all participating subjects gave informed consent.
All peptides: MAP106c121–132 [PGRRPFTRKELQ]; EBNA1400–413 [PGRRPFFHPVGEAD]; MBP85–98 [ENPVVNFFKNIVTP]; and HERV-Wenv73–88 [NPSCPGGLGVTVCWTY] were synthesized at > 90% purity commercially (LifeTein, South Plainfield, NJ 07080, USA).
Wells were coated with 10 μg/ml of peptides dissolved in 50 mM carbonate/bicarbonate buffer and incubated at 4 °C overnight. The day after, 200 μl of blocking solution (PBS-Tween containing 5% milk) was added to each well and the plate was further incubated at room temperature for 1 h. After rinsing with PBS-T, serum samples were added at 1:100 dilutions in PBS-T for 2 h at room temperature. After five washing with PBS-T, alkaline phosphatase-conjugated goat antihuman IgG polyclonal Ab (1:1000) was added for 1 h at room temperature. Finally, after five washing with PBS-T, para-nitrophenylphosphate substrate solution was added to each well and the plates were incubated at room temperature in the dark for 5, 10 and 15 min. The optical density (OD) was read at a 405 nm wavelength using VersaTunable MAX microplate reader. Data was normalized to a positive control serum included in all experiments, the reactivity of which was set at 10.000 arbitrary units (AU)/ml.
2.4. Statistical analysis
Statistics were carried out using GraphPad Prism 6.0 software (San Diego, CA, USA). Continuous variables are presented as mean ± standard deviation (SD), categorical variables as numbers and percentages. MS patients followed during the two years of the study were compared by T student test and repeated measures analysis of variance (rANOVA). T Student test was used to compare T0 and T24 and also T24 and post-natalizumab. The cut-off values were determined using the control group through the ROC curve, and seropositivity rates were calculated according to the cut-off.
The humoral response and Ab seropositivity against EBNA1400–413, MAP106c121–132, MBP85–98 and HERV-Wenv73–88 were evaluated in all MS patients over time. Concerning EBNA1400–413, no statistically significant difference was observed in the antibody level during natalizumab exposure. rANOVA test among the groups T0, T3, T6, and T12 was applied to assess the antibody response over time (p-value 0.5067), therefore no change after 12 months was observed. T student test was applied between T0 and T24 (p-value 0.0662), but it was not significant. T student was also applied to compare T24 and post-NTZ observing that the two groups were statistically similar (p-value 0.1229). Ab seropositivity was considered according to the cut-off value (0.26) determined by ROC; 55% were positive at T0, 52% at T3, 53% at T6, at T12 44%, at T24 50% and 48% were positive in the post-natalizumab group ( Fig. 1 A).
MAP 106c121–132 as well did not show statistically significant differences over time. Similarly rANOVA test among the groups T0, T3, T6, and T12 was applied; no change after 12 months was observed (p-value 0.561). T student test was applied between T0 and T24, showing no significance (p-value 0.0554). T student was also applied to compare T24 and post-NTZ observing that the two groups were statistically similar (p-value 0.1097). Ab seropositivity was calculated according to the AUC in the ROC analysis, cut-off value (0.30); the percentage of positivity was at T0 51%, at T3 46%, at T6 55%, at T12 46%, at T24 25% and in the post-natalizumab 48% ( Fig. 1 B). Similarly, MBP(85–98) did not produce any statistically significant difference over time. rANOVA test between the groups T0, T3, T6, and T12 was applied (p-value 0.8728). T student test was applied between T0 and T24 (p-value 0.268). T student was also applied to compare T24 and post-NTZ observing that the two groups were statistically similar (p-value 0.1235). Cut-off value was calculated at 0.32 according to the ROC analysis; the percentage of positivity at T0 was 36%, at T3 33%, at T6 28%, at T12 31%, at T24 17% and in the post-natalizumab group 37% ( Fig. 1 C). Conversely HERV-Wenv (73–88) showed a statistically significant difference over time during natalizumab exposure and in the post-natalizumab group after two year treatment. rANOVA test between the groups T0, T3, T6, and T12 did not show changes after 12 months (p-value 0.946). T student test was applied between T0 and T24 (p-value 0.0025), and also between T0 and post-NTZ (p-value 0.0008) showing a significant reduction of the antibody titer. The T student was also applied to compare T24 and post-NTZ observing that the two groups were statistically similar (p-value 0.361). The percentage of positivity was at T0 40%, at T3 44%, at T6 43%, at T12 41%, at T24 0% and in the post-natalizumab group 7% ( Fig. 1 D).
Natalizumab induces a continuous decrease of α4 expression  that results in a reduced transmigration of inflammatory cells across the blood– brain barrier (BBB) into the central nervous system (CNS). We set up a study to observe the humoral response against two infectious agents previously associated to MS, EBV and MAP  , and a MS related autoantigen  and the humoral response against HERV-Wenv73–88 whose expression was reported to be modulated during natalizumab therapy  . A recent study has shown an inhibitory effect of natalizumab on surrogates of B cell function in blood and CSF  . We supposed that the drug may be implicated in the decrease of IgG synthesis, as shown in a cross-sectional study of MS patients treated or not with natalizumab  We have observed a significant reduction of the Ab level against HERV-Wenv73–88 in 12 MS patients after 24 month and 27 MS patients after two years of natalizumab treatment. This could depend by the fact that reduced transmigration of the lymphocytes across the CNS may prevents the recognition of the HERV-W env, espressed in the CNS  but it is not supported by the results against the other peptides where was not observed any Ab positivity decrease. Arru et al.  reported a reduced expression of HERV-Wenv after 6 month of natalizumab treatment and this could explain our observations regarding the reduction of the antibody response after two years. The fact that the level decreased only after two years could depend by the fact that the antibody response has a longer dynamics than the decrease of retrovirus expression. Finally, the reduced expression of HERV-W may be due to a decrease of the inflammatory milieu in the CNS after natalizumab treatment and not to a specific action of the monoclonal antibody against the endogenous retrovirus. This effect has not been observed in EBV and MAP responses probably because these infectious agents give a persistent infection and the source of the autoimmunity trigger is not within the CNS as supposed in a study carried out on a Swedish cohort of MS patients treated with natalizumab  .
Our findings suggest that natalizumab treatment reduces Ab production against HERV-Wenv after almost two years of treatment and this could represent a biomarker during natalizumab therapy.
Conflict of interest
The authors have no conflicting financial interests.
This investigation was supported by grants from the Sardinian Region L.R.7 2010 (CRP-25160), Fondazione Banco di Sardegna 2014, and FISM — Fondazione Italiana Sclerosi Multipla (grant number Cod. 2009/R/25).
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a Dipartimento di Scienze Biomediche, Sezione di Microbiologia, Università di Sassari, Viale San Pietro 43b, 07100 Sassari, Italy
b Dipartimento di Medicina Clinica e Sperimentale, Università di Sassari, Viale San Pietro 43b, 07100 Sassari, Italy
⁎ Corresponding author at: Dipartimento di Scienze Biomediche, Università di Sassari, Viale San Pietro 43b, 07100 Sassari, Italy.
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