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Background: Infection with human herpesvirus 6A (HHV-6A) has been suggested to increase multiple sclerosis (MS) risk. However, potential interactions between HHV-6A and environmental/lifestyle risk factors for MS have not previously been studied. Methods: We used two Swedish population-based case-control studies comprising 5993 cases and 5995 controls. Using logistic regression models, subjects with different HHV-6A antibody levels, environmental exposures, and lifestyle habits were compared regarding MS risk, by calculating odds ratios (ORs) with 95% confidence intervals (CIs). Potential interactions between high HHV-6A antibody levels and common environmental exposures and lifestyle factors were evaluated on the additive scale. Results: High HHV-6A antibody levels were associated with increased risk of developing MS (OR = 1.5, 95% CI = 1.4–1.6). Regarding MS risk, significant interactions were observed between high HHV-6A antibody levels and both smoking (attributable proportion (AP) = 0.2, 95% CI = 0.1–0.3), low ultraviolet radiation (UVR) exposure (AP = 0.3, 95% CI = 0.1–0.4), and low vitamin D levels (AP = 0.3, 95% CI = 0.0–0.6). Conclusion: High HHV-6A antibody levels are associated with increased MS risk and act synergistically with common environmental/lifestyle risk factors for MS. Further research is needed to investigate potential mechanisms underlying the interactions presented in this study.

Objective: Novel, highly effective disease-modifying therapies have revolutionized multiple sclerosis (MS) care. However, evidence from large comparative studies on important safety outcomes, such as cancer, is still lacking. Methods: In this nationwide register-based cohort study, we linked data from the Swedish MS register to the Swedish Cancer Register and other national health care and census registers. We included 4,187 first-ever initiations of rituximab, 1,620 of fingolimod, and 1,670 of natalizumab in 6,136 MS patients matched for age, sex, and location to 37,801 non-MS general population subjects. Primary outcome was time to first invasive cancer. Results: We identified 78 invasive cancers among treated patients: rituximab 33 (incidence rate [IR] per 10,000 person-years = 34.4, 95% confidence interval [CI] = 23.7–48.3), fingolimod 28 (IR = 44.0, 95% CI = 29.2–63.5), and natalizumab 17 (IR = 26.0, 95% CI = 15.1–41.6). The general population IR was 31.0 (95% CI = 27.8–34.4). Adjusting for baseline characteristics, we found no difference in risk of invasive cancer between rituximab, natalizumab, and the general population but a possibly higher risk with fingolimod compared to the general population (hazard ratio [HR] = 1.53, 95% CI = 0.98–2.38) and rituximab (HR = 1.68, 95% CI = 1.00–2.84). Interpretation: In this first large comparative study of 3 highly effective MS disease-modifying therapies, no increased risk of invasive cancer was seen with rituximab and natalizumab, compared to the general population. However, there was a borderline-significant increased risk with fingolimod, compared to both the general population and rituximab. It was not possible to attribute this increased risk to any specific type of cancer, and further studies are warranted to validate these findings.

A significant proportion of multiple sclerosis (MS) patients treated with interferon beta-1a (Rebif™) develop anti-drug antibodies (ADA) with a negative impact on treatment efficacy. We hypothesized that high-throughput B-cell receptor (BCR) repertoire analysis could be used to predict and monitor ADA development. To study this we analyzed 228 peripheral blood samples from 68 longitudinally followed patients starting on interferon beta-1a. Our results show that whole blood BCR analysis does not reflect, and does not predict ADA development in MS patients treated with interferon beta-1a. We propose that BCR analysis of phenotypically selected cell subsets or tissues might be more informative.

Interferon beta (IFNβ) is a first line therapy for treatment of multiple sclerosis (MS). However, up to 47% of treated patients will develop neutralizing anti-drug antibodies (NAbs) against IFNβ, which at high titres can inhibit the therapeutic effect of the drug. This study aimed to determine the frequency of transient and fluctuating NAb positivity in a real-world clinical routine setting using a large retrospective international cohort of IFNβ-treated MS patients collected as a part of the ABIRISK consortium (n = 9657). Transient and fluctuating NAbs were rare (2.6% and 0.9%, respectively), but bring noteworthy considerations about clinical decisions in context of NAbs.

Mobility disability (MD) refers to substantial limitations in life activities that arise because of movement impairments. Although MD is most prevalent in older individuals, it can also affect younger adults. Increasing evidence suggests that inflammation can drive the development of MD and may need to be targeted for MD prevention. Physical exercise has anti-inflammatory properties and has been associated with MD prevention. However, no studies to date have examined whether exercise interventions affect the peripheral inflammatory status in younger adults with MD. To this end, we used blood samples from young and middle-aged adults with MD (N = 38; median age = 34 years) who participated in a 12-week intervention that included aerobic and resistance exercise training. A pre-post assessment of inflammatory biomarkers was conducted in plasma from two timepoints, i.e., before the exercise trial and at follow-up (3–7 days after the last exercise session). We successfully measured 15 inflammatory biomarkers and found that exercise was associated with a significant reduction in levels of soluble fractalkine, transforming growth factor beta 1 (TGF-β1), eotaxin-1 and interleukin (IL) 6 (corrected α = 0.004). We also found significant male-specific effects of exercise on (i) increasing IL-16 and (ii) decreasing vascular endothelial growth factor-A (VEGF-A). In line with our results, previous studies have also found that exercise can reduce levels of TGF-β1, eotaxin-1 and IL-6. However, our finding that exercise reduces plasma levels of fractalkine in younger adults with MD, as well as the sex-dependent findings, have not been previously reported and warrant replication in larger cohorts. Given the suggested role of inflammation in promoting MD development, our study provides additional support for the use of physical exercise as a treatment modality for MD.

Objective: To estimate risks for all-cause mortality and for severe COVID-19 in multiple sclerosis patients and across relapsing-remitting multiple sclerosis patients exposed to disease-modifying therapies. Methods: We conducted a Swedish nationwide population-based multi-register linkage cohort study and followed all multiple sclerosis patients (n = 17,692 in March 2020), individually age-, sex-, and region-matched to five population-based controls (n = 86,176 in March 2020) during March 2020-June 2021. We compared annual all-cause mortality within and across cohorts, and assessed incidence rates and relative risks for hospitalization, intensive care admission, and death due to COVID-19 in relation to disease-modifying therapy use, using Cox regression. Results: Absolute all-cause mortality among multiple sclerosis patients was higher from March to December 2020 than in previous years, but relative risks versus the population-based controls were similar to preceding years. Incidence rates of hospitalization, intensive care admission, and death due to COVID-19 remained in line with those for all-cause hospitalization, intensive care admission, and mortality. Among relapsing-remitting patients on rituximab, trends for differences in risk of hospitalization due to COVID-19 remained in the demographics-, socioeconomic status-, comorbidity-, and multiple sclerosis severity-adjusted model. Interpretation: Risks of severe COVID-19-related outcomes were increased among multiple sclerosis patients as a whole compared to population controls, but risk increases were also seen for non-COVID-19 hospitalization, intensive care admission, and mortality, and did not significantly differ during the pandemic compared to pre-pandemic years. The risk conveyed by disease-modifying therapies was smaller than previously assumed, likely as a consequence of the possibility to better control for confounders. Funding Agencies|Patient-Centered Outcomes Research Institute (PCORI) Award [MS-1511-33196]; Swedish MRC [2020-02700]; Swedish Research Council [2021-01418]; Swedish Research Council for Health, Working Life, and Welfare [2020-0115]; Knut and Alice Wallenbergs foundation [KAW 2020.0299_VC-2020-0040/VC-2021-0018]

B cell abnormalities are common in systemic lupus erythematosus (SLE), and include expansion of double negative (DN) and age-associated-like B cells (ABC-like). We aimed to investigate rituximab (RTX) effects on DN and ABC-like B-cell subsets and, when possible, also secondary effects on T cells. Fifteen SLE patients, fulfilling the ACR 1982 criteria, starting RTX and followed longitudinally up to two years, were analyzed for B- and T- lymphocyte subsets using multicolor flow cytometry. DN were defined as IgD-CD27- and ABC-like as CD11c+CD21- within the DN gate. Additional phenotyping was performed adding CXCR5 in the B-cell panel. Cellular changes were further analyzed in the context of the generation of anti-drug antibodies (ADA) against RTX and clinical information. The SLE patients were mainly females (86.6%), of median age 36.7 (29.8-49.4) years and disease duration of 6.1 (1.6-11.8) years. Within the DN subset, ABC-like (IgD-CD27-CD11c+CD21-) B cell frequency reduced from baseline median level of 20.4% to 11.3% (p=0.03), at early follow-up. The DN B cells were further subdivided based on CXCR5 expression. Significant shifts were observed at the early follow-up in the DN2 sub-cluster (CD11c+CXCR5-), which reduced significantly (-15.4 percentage points, p=0.02) and in the recently described DN3 (CD11c-CXCR5-) which increased (+13 percentage points, p=0.03). SLE patients treated with RTX are at high risk of developing ADA. In our cohort, the presence of ADA at 6 months was associated with lower frequencies of DN cells and to a more pronounced expansion of plasmablasts at early follow-up. The frequency of follicular helper T cells (T

Objective: Infliximab-treated patients with rheumatoid arthritis (RA) may respond insufficiently due to low serum infliximab (sIFX) levels, caused by anti-drug antibodies (ADAs). However, monitoring of sIFX and ADAs is not routinely implemented, and levels for optimal outcome have not been validated. We searched for predictors for sIFX 0.2 μg/mL]. The primary and secondary outcome measures were low disease activity [LDA = 28-joint Disease Activity Score (DAS28) ≤ 3.2] and remission (DAS28 7.0 μg/mL showed a dose–response association with LDA (30%, 64%, 67%, and 79%, respectively, p = 0.008) and remission (10%, 45%, 39%, and 66%, p = 0.004) at trial cessation (21 months). Female patients had sIFX 0.2 μg/mL, of whom three were ADA positive at other time-points, all with high DAS28 at follow-up. Conclusion: In early RA patients receiving IFX as a second-line agent, sIFX

Patients with chronic inflammatory diseases are often treated with immunosuppressants and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological tests will improve our understanding of the infection and immunity in this population, unless they tests give false positive results. The aim of this study was to evaluate the specificity of SARS-Cov-2 serological assays using samples from patients with chronic inflammatory diseases collected prior to April 2019, thus defined as negative. Samples from patients with multiple sclerosis (MS, n=10), rheumatoid arthritis (RA, n=47) with or without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-CCP2) and systemic lupus erythematosus (SLE, n=10) with or without RF, were analyzed for SARS-CoV-2 antibodies using 17 commercially available lateral flow assays (LFA), two ELISA kits and one in-house developed IgG multiplex bead-based assay. Six LFA and the in-house validated IgG assay correctly produced negative results for all samples. However, the majority of assays (n=13), gave false positive signal for samples from patients with RA and SLE. This was most notable in samples from RF positive RA patients. No false positive samples were detected in any assay using samples from patients with MS. Poor specificity of commercial serological assays could possibly be, at least partly, due to interfering antibodies in samples from patients with chronic inflammatory diseases. For these patients, the risk of false positivity should be considered when interpreting results of the SARS-CoV-2 serological assays.

Background and purpose Epstein-Barr virus (EBV) and human herpesvirus 6A (HHV-6A) are associated with increased risk of multiple sclerosis (MS). Conversely, infection with cytomegalovirus (CMV) has been suggested to reduce the risk of MS but supporting data from presymptomatic studies are lacking. Here, it was sought to increase the understanding of CMV in MS aetiology. Methods A nested case-control study was performed with presymptomatically collected blood samples identified through crosslinkage of MS registries and Swedish biobanks. Serological antibody response against CMV, EBV and HHV-6A was determined using a bead-based multiplex assay. Odds ratio (OR) with 95% confidence interval (CI) for CMV seropositivity as a risk factor for MS was calculated by conditional logistic regression and adjusted for EBV and HHV-6A seropositivity. Potential interactions on the additive scale were analysed by calculating the attributable proportion due to interaction (AP). Results Serum samples from 670 pairs of matched cases and controls were included. CMV seropositivity was associated with a reduced risk for MS (OR = 0.70, 95% CI 0.56-0.88, p = 0.003). Statistical interactions on the additive scale were observed between seronegativity for CMV and seropositivity against HHV-6A (AP 0.34, 95% CI 0.06-0.61) and EBV antigen EBNA-1 (amino acid 385-420) at age 20-39 years (AP 0.37, 95% CI 0.09-0.65). Conclusions Cytomegalovirus seropositivity is associated with a decreased risk for MS. The protective role for CMV infection in MS aetiology is further supported by the interactions between CMV seronegativity and EBV and HHV-6A seropositivity. Funding Agencies|Swedish Research CouncilSwedish Research CouncilEuropean Commission [2015-02419, 2018-03918]; Visare Norr Fund, Northern County Councils Regional Federation [940405]; Research and Development Unit, Region Jamtland Harjedalen [JLL-939768]; Margaretha af Ugglas stiftelse; Swedish Neuro Foundation; MS Research fund; Swedish Research CouncilSwedish Research CouncilEuropean Commission; Swedish Brain Foundation; Horizon 2020 MultipleMS Grant [733161]

Background Anti-drug antibodies (ADAs) can impact on the efficacy and safety of biologicals, today used to treat several chronic inflammatory conditions. Specific patient groups may be more prone to develop ADAs. Rituximab is routinely used for ANCA-associated vasculitis (AAV) and as off-label therapy for systemic lupus erythematosus (SLE), but data on occurrence and predisposing factors to ADAs in these diseases is limited. Objectives To elucidate the rate of occurrence, and risk factors for ADAs against rituximab in SLE and AAV. Methods ADAs were detected using a bridging electrochemiluminescent (ECL) immunoassay in sera from rituximab-naïve (AAV; n = 41 and SLE; n = 62) and rituximab-treated (AAV; n = 22 and SLE; n = 66) patients. Clinical data was retrieved from medical records. Disease activity was estimated by the SLE Disease Activity Index-2000 (SLEDAI-2 K) and the Birmingham Vasculitis Activity Score (BVAS). Results After first rituximab cycle, no AAV patients were ADA-positive compared to 37.8% of the SLE patients. Samples were obtained at a median (IQR) time of 5.5 (3.7–7.0) months (AAV), and 6.0 (5.0–7.0) months (SLE). ADA-positive SLE individuals were younger (34.0 (25.9–40.8) vs 44.3 (32.7–56.3) years, p = 0.002) and with more active disease (SLEDAI-2 K 14.0 (10.0–18.5) vs. 8.0 (6.0–14), p = 0.0017) and shorter disease duration (4.14 (1.18–10.08) vs 9.19 (5.71–16.93), p = 0.0097) compared to ADA-negative SLE. ADAs primarily occurred in nephritis patients, were associated with anti-dsDNA positivity but were not influenced by concomitant use of corticosteroids, cyclophosphamide or previous treatments. Despite overall reduction of SLEDAI-2 K (12.0 (7.0–16) to 4.0 (2.0–6.7), p p = 0.004) and their B cell count at 6 months follow-up was higher (CD19 + % 4.0 (0.5–10.0) vs 0.5 (0.4–1.0), p = 0.002). At retreatment, two ADA-positive SLE patients developed serum sickness (16.7%), and three had infusion reactions (25%) in contrast with one (5.2%) serum sickness in the ADA-negative group. Conclusions In contrast to AAV, ADAs were highly prevalent among rituximab-treated SLE patients already after the first course of treatment and were found to effect on both clinical and immunological responses. The high frequency in SLE may warrant implementations of ADA screening before retreatment and survey of immediate and late-onset infusion reactions.

Background With the growth in use of biotherapic drugs in various medical fields, the occurrence of anti-drug antibodies represents nowadays a serious issue. This immune response against a drug can be due either to pre-existing antibodies or to the novel production of antibodies from B-cell clones by a fraction of the exposed subjects. Identifying genetic markers associated with the immunogenicity of biotherapeutic drugs may provide new opportunities for risk stratification before the introduction of the drug. However, real-world investigations should take into account that the population under study is a mixture of pre-immune, immune-reactive and immune-tolerant subjects. Method In this work, we propose a novel test for assessing the effect of genetic markers on drug immunogenicity taking into account that the population under study is a mixed one. This test statistic is derived from a novel two-part semiparametric improper survival model which relies on immunological mechanistic considerations. Results Simulation results show the good behavior of the proposed statistic as compared to a two-part logrank test. In a study on drug immunogenicity, our results highlighted findings that would have been discarded when considering classical tests. Conclusion We propose a novel test that can be used for analyzing drug immunogenicity and is easy to implement with standard softwares. This test is also applicable for situations where one wants to test the equality of improper survival distributions of semi-continuous outcomes between two or more independent groups.

Background Biopharmaceutical products (BPs) are widely used to treat autoimmune diseases, but immunogenicity limits their efficacy for an important proportion of patients. Our knowledge of patient-related factors influencing the occurrence of antidrug antibodies (ADAs) is still limited. Methods and findings The European consortium ABIRISK (Anti-Biopharmaceutical Immunization: prediction and analysis of clinical relevance to minimize the RISK) conducted a clinical and genomic multicohort prospective study of 560 patients with multiple sclerosis (MS, n = 147), rheumatoid arthritis (RA, n = 229), Crohn’s disease (n = 148), or ulcerative colitis (n = 36) treated with 8 different biopharmaceuticals (etanercept, n = 84; infliximab, n = 101; adalimumab, n = 153; interferon [IFN]-beta-1a intramuscularly [IM], n = 38; IFN-beta-1a subcutaneously [SC], n = 68; IFN-beta-1b SC, n = 41; rituximab, n = 31; tocilizumab, n = 44) and followed during the first 12 months of therapy for time to ADA development. From the bioclinical data collected, we explored the relationships between patient-related factors and the occurrence of ADAs. Both baseline and time-dependent factors such as concomitant medications were analyzed using Cox proportional hazard regression models. Mean age and disease duration were 35.1 and 0.85 years, respectively, for MS; 54.2 and 3.17 years for RA; and 36.9 and 3.69 years for inflammatory bowel diseases (IBDs). In a multivariate Cox regression model including each of the clinical and genetic factors mentioned hereafter, among the clinical factors, immunosuppressants (adjusted hazard ratio [aHR] = 0.408 [95% confidence interval (CI) 0.253–0.657], p < 0.001) and antibiotics (aHR = 0.121 [0.0437–0.333], p < 0.0001) were independently negatively associated with time to ADA development, whereas infections during the study (aHR = 2.757 [1.616–4.704], p < 0.001) and tobacco smoking (aHR = 2.150 [1.319–3.503], p < 0.01) were positively associated. 351,824 Single-Nucleotide Polymorphisms (SNPs) and 38 imputed Human Leukocyte Antigen (HLA) alleles were analyzed through a genome-wide association study. We found that the HLA-DQA1*05 allele significantly increased the rate of immunogenicity (aHR = 3.9 [1.923–5.976], p < 0.0001 for the homozygotes). Among the 6 genetic variants selected at a 20% false discovery rate (FDR) threshold, the minor allele of rs10508884, which is situated in an intron of the CXCL12 gene, increased the rate of immunogenicity (aHR = 3.804 [2.139–6.764], p < 1 × 10−5 for patients homozygous for the minor allele) and was chosen for validation through a CXCL12 protein enzyme-linked immunosorbent assay (ELISA) on patient serum at baseline before therapy start. CXCL12 protein levels were higher for patients homozygous for the minor allele carrying higher ADA risk (mean: 2,693 pg/ml) than for the other genotypes (mean: 2,317 pg/ml; p = 0.014), and patients with CXCL12 levels above the median in serum were more prone to develop ADAs (aHR = 2.329 [1.106–4.90], p = 0.026). A limitation of the study is the lack of replication; therefore, other studies are required to confirm our findings. Conclusion In our study, we found that immunosuppressants and antibiotics were associated with decreased risk of ADA development, whereas tobacco smoking and infections during the study were associated with increased risk. We found that the HLA-DQA1*05 allele was associated with an increased rate of immunogenicity. Moreover, our results suggest a relationship between CXCL12 production and ADA development independent of the disease, which is consistent with its known function in affinity maturation of antibodies and plasma cell survival. Our findings may help physicians in the management of patients receiving biotherapies., In a multicohort prospective study of patients from 12 countries, Signe Hässler and colleagues investigate clinical and genetic factors associated with development of anti-biopharmaceutical drug antibodies in patients with autoimmune diseases., Author summary Why was this study done? Biopharmaceutical products such as monoclonal antibodies are widely used to treat autoimmune diseases. Biopharmaceutical products may induce the development of antidrug antibodies, which often lead to therapy failure. Patient-related factors that influence the development of antidrug antibodies need to be characterized. What did the researchers do and find? We set up a European multicohort prospective study on 4 autoimmune diseases (multiple sclerosis, rheumatoid arthritis, Crohn’s disease, and ulcerative colitis) treated with 8 different biopharmaceutical products. We collected demographic and clinical data and tested antidrug antibodies in longitudinal serum samples from 560 patients. For 457 patients who gave consent, we also collected genetic data. We identified antibiotics and immunosuppressants as negatively associated risk factors and heavy smoking, infections during the study, the HLA-DQA1*05 allele and a minor variant in the CXCL12 chemokine gene associated with increased protein expression as risk factors of antidrug antibody development. What do these findings mean? Our findings suggest that the combination of immunosuppressant and biopharmaceutical therapy could be associated with decreased risk of antidrug antibody occurrence, which has implications for rheumatoid arthritis and inflammatory bowel diseases, for which immunosuppressants are often, but not always, given together with biopharmaceuticals. Patients heterozygotes or homozygotes for the HLA-DQA1*05 allele may have an increased risk of antidrug antibody occurrence associated with biopharmaceutical therapy. The small study size warrants a validation through independent studies, in particular for the genetic findings.

Background Neutralizing anti-drug antibodies (NAbs) to interferon beta (IFNβ) develop in up to 47% of multiple sclerosis (MS) treated patients inhibiting treatment effect of IFNβ. However, the long-term effect of NAbs remain unknown. Objective To investigate the long-term consequences of high titer NAbs to IFNβ on disease activity and progression in MS patients. Methods An observational study including data from all IFNβ treated relapsing remitting MS patients with sufficient NAb test results from the Swedish MS registry. Patients were classified into either confirmed ‘high titer’ or ‘persistent negative’ groups and analyzed for differences in disease activity and progression over time. Results A total of 197 high-titer and 2907 persistent negative patients with 19969.6 follow up years of data were included. High titer NAbs were associated with a higher degree of disease activity at baseline. However, even when accounting for this, the presence of high titer NAbs were also associated with higher disease activity during IFNβ treatment. This persisted even after the next DMT start, suggesting that earlier high titers may partially reduce the effect of later treatments. No difference was found in confirmed disability progression. Conclusion High titer NAbs to IFNβ are associated with higher disease activity, persisting even after IFNβ discontinuation or switch. These results support use of highly efficient treatment earlier in patients with active disease, to avoid these complications.

Background: Neutralizing anti-drug antibodies (ADA) can greatly reduce the efficacy of biopharmaceuticals used to treat patients with multiple sclerosis (MS). However, the biological factors pre-disposing an individual to develop ADA are poorly characterized. Thus, there is an unmet clinical need for biomarkers to predict the development of immunogenicity, and subsequent treatment failure. Up to 35% of MS patients treated with beta interferons (IFNβ) develop ADA. Here we use machine learning to predict immunogenicity against IFNβ utilizing serum metabolomics data. Methods: Serum samples were collected from 89 MS patients as part of the ABIRISK consortium-a multi-center prospective study of ADA development. Metabolites and ADA were quantified prior to and after IFNβ treatment. Thirty patients became ADA positive during the first year of treatment (ADA+). We tested the efficacy of six binary classification models using 10-fold cross validation; k-nearest neighbors, decision tree, random forest, support vector machine and lasso (Least Absolute Shrinkage and Selection Operator) logistic regression with and without interactions. Results: We were able to predict future immunogenicity from baseline metabolomics data. Lasso logistic regression with/without interactions and support vector machines were the most successful at identifying ADA+ or ADA- cases, respectively. Furthermore, patients who become ADA+ had a distinct metabolic response to IFNβ in the first 3 months, with 29 differentially regulated metabolites. Machine learning algorithms could also predict ADA status based on metabolite concentrations at 3 months. Lasso logistic regressions had the greatest proportion of correct classifications [F1 score (accuracy measure) = 0.808, specificity = 0.913]. Finally, we hypothesized that serum lipids could contribute to ADA development by altering immune-cell lipid rafts. This was supported by experimental evidence demonstrating that, prior to IFNβ exposure, lipid raft-associated lipids were differentially expressed between MS patients who became ADA+ or remained ADA-. Conclusion: Serum metabolites are a promising biomarker for prediction of ADA development in MS patients treated with IFNβ, and could provide novel insight into mechanisms of immunogenicity.

Two validated assays, a bridging ELISA and a luciferase-based bioassay, were compared for detection of anti-drug antibodies (ADA) against interferon-beta (IFN-β) in patients with multiple sclerosis. Serum samples were tested from patients enrolled in a prospective study of 18 months. In contrast to the ELISA, when IFN-β-specific rabbit polyclonal and human monoclonal antibodies were tested, the bioassay was the more sensitive to detect IFN-β ADA in patients' sera. For clinical samples, selection of method of ELISA should be evaluated prior to the use of a multi-tiered approach. A titer threshold value is reported that may be used as a predictor for persistently positive neutralizing ADA.

International audience; Predicting immunogenicity for biotherapies using patient and drug-related factors represents nowadays a challenging issue. With the growing ability to collect massive amount of data, machine learning algorithms can provide efficient predictive tools. From the bio-clinical data collected in the multi-cohort of autoimmune diseases treated with biotherapies from the ABIRISK consortium, we evaluated the predictive power of a custom-built random survival forest for predicting the occurrence of anti-drug antibodies. This procedure takes into account the existence of a population composed of immune-reactive and immune-tolerant subjects as well as the existence of a tiny expected proportion of relevant predictive variables. The practical application to the ABIRISK cohort shows that this approach provides a good predictive accuracy that outperforms the classical survival random forest procedure. Moreover, the individual predicted probabilities allow to separate high and low risk group of patients. To our best knowledge, this is the first study to evaluate the use of machine learning procedures to predict biotherapy immunogenicity based on bioclinical information. It seems that such approach may have potential to provide useful information for the clinical practice of stratifying patients before receiving a biotherapy.

Background Human herpesvirus 6B (HHV-6B) is a neurotropic virus that has been repeatedly associated with mesial temporal lobe epilepsy (MTLE). However, the mechanism behind this suggested association is not known. Therefore, the aim of this study was to investigate what genes were affected by HHV-6B, possibly revealing HHV-6B induced disease causing mechanisms. Material and method First, gene expression in MTLE tissue positive for HHV-6B DNA (n = 10) and negative for HHV-6B DNA (n = 14) was compared using the Affymetrix® Human Gene 2.1 ST Array. Secondly, in vitro experiments were conducted where Molt-3 T cells were infected with HHV-6B and gene expression of MAP2K4 (MKK4) and 89 other genes in the MAPK signaling pathway was investigated using qPCR. In addition, phosphorylated MKK4 was assessed using IFA and the DNA methylation investigated with Illumina Infinium HumanMethylation450 BeadChip array. Results MAP2K4 was one of the most differently expressed genes in the Affymetrix array, suggesting an upregulation by HHV-6B infection in MTLE tissue. No gene reached statistical significance but MAP2K4 was selected for further investigation in vitro, where it was clearly upregulated by HHV-6B infection both on gene expression and protein expression level. Further investigating expression of genes in the MAPK pathways in vitro revealed that several genes were affected by HHV-6B infection, but none of these genes displayed viral induced changes in DNA methylation. Conclusions As the MAPK pathways are involved in transforming different stimuli (like stress) into a cellular responses (like apoptosis or inflammation), it may not be surprising that genes in these pathways are affected by virus infection. This is the first report of HHV-6B’s effect on these signaling cascades and given that both dysregulation of the MAPK pathways and an association with HHV-6B have been previously observed in epilepsy, a possible link of infection induced dysregulation of MAPK in epilepsy warrant further investigation.

International audience; OBJECTIVES:To evaluate the incidence of anti-drug antibody (ADA) occurrences and ADA-related risk factors under adalimumab and infliximab treatment in rheumatoid arthritis (RA) patients.METHODS:The study combined retrospective cohorts from the ABIRISK project totaling 366 RA patients treated with adalimumab (n = 240) or infliximab (n = 126), 92.4% of them anti-TNF naive (n = 328/355) and 96.6% of them co-treated with methotrexate (n = 341/353) with up to 18 months follow-up. ADA positivity was measured by enzyme-linked immunosorbent assay. The cumulative incidence of ADA was estimated, and potential bio-clinical factors were investigated using a Cox regression model on interval-censored data.RESULTS:ADAs were detected within 18 months in 19.2% (n = 46) of the adalimumab-treated patients and 29.4% (n = 37) of the infliximab-treated patients. The cumulative incidence of ADA increased over time. In the adalimumab and infliximab groups, respectively, the incidence was 15.4% (5.2-20.2) and 0% (0-5.9) at 3 months, 17.6% (11.4-26.4) and 0% (0-25.9) at 6 months, 17.7% (12.6-37.5) and 34.1% (11.4-46.3) at 12 months, 50.0% (25.9-87.5) and 37.5% (25.9-77.4) at 15 months and 50.0% (25.9-87.5) and 66.7% (37.7-100) at 18 months. Factors associated with a higher risk of ADA development were: longer disease duration (1-3 vs.

Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the "Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk" (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519 ng/mL for LUCIMU, 814 ng/mL for LUCRH, and 320 ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low positive titers needs to be further evaluated.

Interferon beta (IFNβ) is used as a first-line treatment for multiple sclerosis (MS) and is injected intramuscularly or subcutaneously (s.c.). The subcutaneous route is considered more immunogenic as it is associated with increased antidrug antibody-positive patients. The skin contains dendritic cells (DCs) and it is unclear whether these contribute to immunogenicity. To assess the effect of IFNβ on skin-resident cells, IFNβ was injected intradermally (i.d.) ex vivo using a human skin explant model or s.c. in vivo in MS patients. Ex vivo, intradermal IFNβ injections reduced migration and enhanced surface CD86 expression of dermal DCs, and an increased expression of HLA-DR+ was observed in skin biopsies taken after subcutaneous IFNβ injection (in vivo). In both models, IFNβ elevated the expression of several inflammatory cytokines when compared to the control biopsies. Our results show that 3 different IFNβ preparations, normalized in dose and injection site, induce similar immune responses, suggesting that the differences in immunogenicity are likely due to the route and frequency of administration.

NK cells play an important role in the defense against viral infections. However, little is known about the regulation of NK cell responses during the first days of acute viral infections in humans. In this study, we used the live attenuated yellow fever virus (YFV) vaccine 17D as a human in vivo model to study the temporal dynamics and regulation of NK cell responses in an acute viral infection. YFV induced a robust NK cell response in vivo, with an early activation and peak in NK cell function at day 6, followed by a delayed peak in Ki67 expression, which was indicative of proliferation, at day 10. The in vivo NK cell response correlated positively with plasma type I/III IFN levels at day 6, as well as with the viral load. YFV induced an increased functional responsiveness to IL-12 and IL-18, as well as to K562 cells, indicating that the NK cells were primed in vivo. The NK cell responses were associated primarily with the stage of differentiation, because the magnitude of induced Ki67 and CD69 expression was distinctly higher in CD57− NK cells. In contrast, NK cells expressing self- and nonself-HLA class I–binding inhibitory killer cell Ig-like receptors contributed, to a similar degree, to the response. Taken together, our results indicate that NK cells are primed by type I/III IFN in vivo early after YFV infection and that their response is governed primarily by the differentiation stage, independently of killer cell Ig-like receptor/HLA class I–mediated inhibition or education.

Chronic inflammatory diseases are now treated with a range of different biopharmaceuticals, often requiring lifelong parenteral administrations. This exposure to drugs is unnatural and can trigger the immune system and result in the formation of antidrug antibodies. Drug-specific antibodies will, if of sufficiently high titre and affinity, block the intended effect of the drug, increase its clearance and make continued treatment worthless. We expect the immune system to react towards therapies against which tolerance has never been established, which is the case for factor VIII treatment in patients with haemophilia A. However, even biopharmaceutical molecules that we should be tolerant against can elicit antidrug antibodies, for instance in treatment of multiple sclerosis patients with recombinant human interferon-beta. Possible immunological mechanisms behind the breaking of tolerance against drugs, the impact this has on continuous treatment success, clinical practice and drug development, will be discussed in this review.

Background: Anti-drug antibodies (ADA) against natalizumab develop early during treatment. ADA persistency is defined by two consecutive positive results as performed by the current qualitative ELISA assay (positive/negative). Very little is known about the magnitude of the natalizumab ADA response and persistency. Design/methods: We developed a highly sensitive natalizumab ADA titration assay on the Meso Scale Discovery (MSD) platform and a pharmacokinetic (PK) assay. We included 43 patients with a positive ELISA-ADA result within 6 months of treatment initiation (baseline) of whom a follow-up serum sample was available 12–30 months after treatment start. MSD-ADA titres and drug levels were measured. Results: Median MSD-ADA titre at baseline was 4881 and 303 at follow-up. A titre of >400 at baseline had a 94% sensitivity and 89% specificity to predict ADA persistency. Reversion to ADA negativity occurred in 10 patients with mean drug levels of 10.8 μg/mL. The median trough drug level in ADA-positive samples was 0 µg/mL. PK levels and ADA titres correlated strongly negatively ( r = −0.67). Conclusion: High baseline natalizumab ADA titres accurately predict persistency. Despite continuous treatment, the majority of patients with persistent ADA had no detectable drug levels indicating loss of efficacy in line with phase 3 study results.

Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-β is an established treatment for MS; however, up to 30% of IFN-β-treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14+ monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-β. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-β administration. Reduced monocyte NOTCH2 expression in nADA+ MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA+ patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-β administration.

Purpose Mesial temporal lobe epilepsy (MTLE) is a common epileptic disorder. Although likely multifactorial, the mechanisms underlying the etiology and pathogenesis of the disease remains unknown in majority of patients. Viruses, particularly Human Herpes Virus 6A and B (HHV-6), two neurotropic herpes viruses, have been implicated in MTLE due to their ubiquitous nature and ability to establish lifelong latency with risk of reactivation. However, the results of studies investigating this relationship are conflicting. This systematic review and meta -analysis was conducted to determine the relationship between HHV-6 DNA (not specifying if A or B) in brain tissue and MTLE based on the current evidence. Method Two independent assessors carried out a comprehensive electronic search to identify all relevant studies. Both fixed- and random-effects models were used to determine the overall odds ratio. Results A total of 10 studies met the inclusion criteria for the systematic review and eight for the meta -analysis. In 19.6% of all MTLE patients HHV-6 DNA was detected in brain tissue compared to 10.3% of all controls (p >0.05). The pooled odds ratio of HHV-6 positive cases in MTLE patients was 2.016 [95%-CI: 1.16-3.50] in the fixed effect model. Conclusion The results of this meta -analysis indicate an association between HHV-6 DNA and MTLE surgically resected tissue samples, unspecified if A or B or both. However, the casual relationship and possible pathological role of HHV-6 in MTLE are yet to be elucidated. This study's results provide a basis for future studies continuing the investigation into pathological implications of HHV-6.

Human herpesvirus 6B (HHV-6B) is a neurotropic betaherpesvirus that achieves latency by integrating its genome into host cell chromosomes. Several viruses can induce epigenetic modifications in their host cells, but no study has investigated the epigenetic modifications induced by HHV-6B. This study analyzed methylation with an Illumina 450K array, comparing HHV-6B-infected and uninfected Molt-3 T cells 3 days postinfection. Bisulfite pyrosequencing was used to validate the Illumina results and to investigate methylation over time in vitro . Expression of genes was investigated using quantitative PCR (qPCR), and virus integration was investigated with PCR. A total of 406 CpG sites showed a significant HHV-6B-induced change in methylation in vitro . Remarkably, 86% (351/406) of these CpGs were located IMPORTANCE The ability to establish latency in the host is a hallmark of herpesviruses, but the mechanisms differ. Human herpesvirus 6B (HHV-6B) is known to establish latency through integration of its genome into the telomeric regions of host cells, with the ability to reactivate. Our study is the first to show that HHV-6B specifically induces hypomethylated regions close to the telomeres and that integrating viruses may use the host methylation machinery to facilitate their integration process. The results from this study contribute to knowledge of HHV-6B biology and virus-host interaction. This in turn will lead to further progress in our understanding of the underlying mechanisms by which HHV-6B contributes to pathological processes and may have important implications in both disease prevention and treatment.

IMPORTANCE: Although highly effective disease-modifying therapies for multiple sclerosis (MS) have been associated with an increased risk of infections vs injectable therapies interferon beta and glatiramer acetate (GA), the magnitude of potential risk increase is not well established in real-world populations. Even less is known about infection risk associated with rituximab, which is extensively used off-label to treat MS in Sweden. OBJECTIVE: To examine the risk of serious infections associated with disease-modifying treatments for MS. DESIGN, SETTING, AND PARTICIPANTS: This nationwide register-based cohort study was conducted in Sweden from January 1, 2011, to December 31, 2017. National registers with prospective data collection from the public health care system were used. All Swedish patients with relapsing-remitting MS whose data were recorded in the Swedish MS register as initiating treatment with rituximab, natalizumab, fingolimod, or interferon beta and GA and an age-matched and sex-matched general population comparator cohort were included. EXPOSURES: Treatment with rituximab, natalizumab, fingolimod, and interferon beta and GA. MAIN OUTCOMES AND MEASURES: Serious infections were defined as all infections resulting in hospitalization. Additional outcomes included outpatient treatment with antibiotic or herpes antiviral medications. Adjusted hazard ratios (HRs) were estimated in Cox regressions. RESULTS: A total of 6421 patients (3260 taking rituximab, 1588 taking natalizumab, 1535 taking fingolimod, and 2217 taking interferon beta/GA) were included, plus a comparator cohort of 42 645 individuals. Among 6421 patients with 8600 treatment episodes, the mean (SD) age at treatment start ranged from 35.0 (10.1) years to 40.4 (10.6) years; 6186 patients were female. The crude rate of infections was higher in patients with MS taking interferon beta and GA than the general population (incidence rate, 8.9 [95% CI, 6.4-12.1] vs 5.2 [95% CI, 4.8-5.5] per 1000 person-years), and higher still in patients taking fingolimod (incidence rate, 14.3 [95% CI, 10.8-18.5] per 1000 person-years), natalizumab (incidence rate, 11.4 [95% CI, 8.3-15.3] per 1000 person-years), and rituximab (incidence rate, 19.7 [95% CI, 16.4-23.5] per 1000 person-years). After confounder adjustment, the rate remained significantly higher for rituximab (HR, 1.70 [95% CI, 1.11-2.61]) but not fingolimod (HR, 1.30 [95% CI, 0.84-2.03]) or natalizumab (HR, 1.12 [95% CI, 0.71-1.77]) compared with interferon beta and GA. In contrast, use of herpes antiviral drugs during rituximab treatment was similar to that of interferon beta and GA and lower than that of natalizumab (HR, 1.82 [1.34-2.46]) and fingolimod (HR, 1.71 [95% CI, 1.27-2.32]). CONCLUSIONS AND RELEVANCE: Patients with MS are at a generally increased risk of infections, and this differs by treatment. The rate of infections was lowest with interferon beta and GA; among newer treatments, off-label use of rituximab was associated with the highest rate of serious infections. The different risk profiles should inform the risk-benefit assessments of these treatments.

Background Human herpesvirus 6 (HHV-6) has been reported to be associated with multiple sclerosis (MS) and Guillain–Barre syndrome (GBS). Methods We analyzed cell-free HHV-6 DNA as an indication of active infection in the peripheral blood and cerebrospinal fluid (CSF) of Swedish patients with GBS, patients with chronic inflammatory demyelinating polyradiculoneuropathy, treatment-naive patients with possible MS, interferon-β treated MS patients [with or without neutralizing antibodies (NAbs)], and control patients with headache. Results One of 14 GBS patients and one of eight patients with chronic inflammatory demyelinating polyradiculoneuropathy were positive for HHV-6 DNA in serum. Of the 27 treatment-naive possible MS patients, two were positive in plasma and one in CSF. HHV-6 DNA was detected in the serum of three of 79 NAb+ patients and one of 102 NAb-interferon-β treated MS patients. HHV-6 DNA could not be detected in the plasma or CSF of any of the 33 controls, although the differences were not statistically significant. Conclusion Our results do not suggest active HHV-6 infection to be a common phenomenon in any of the patient groups studied.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system. Both genetic and environmental factors contribute to disease susceptibility and two viruses associated with MS are human herpesvirus (HHV)-6A and HHV-6B, together referred to as HHV-6. This study characterized the plasma IgG antibody response against HHV-6 in MS patients (n=446) and healthy controls (n=487), and the relationship between MS susceptibility factors and the anti-HHV-6 response was investigated. In addition, 134 samples were further investigated for IgG against the early HHV-6 antigen p41. Antibody levels were measured with ELISA. The overall seroprevalence against HHV-6 was 90%, with no significant difference in positivity or levels between MS patients and controls. Interestingly, carriership of HLA-A(∗)02 and tobacco smoking was associated with lower anti-HHV-6 IgG levels (p=0.0017 and p=0.026 respectively), whereas females sex was associated with higher levels (p=0.0090). No difference in IgG titers against p41 was observed between MS patients and controls. In conclusion, the IgG response against HHV-6 was associated with several factors that have previously been associated with MS susceptibility, possibly reflecting a relation between autoimmunity and how the immune system handles viral infections.

Objective: To assess changes in the T-cell receptor (TCR) repertoire in peripheral venous blood and CSF of patients with multiple sclerosis (MS) treated with natalizumab and the potential implication for developing progressive multifocal leukoencephalopathy (PML) and PML–immune reconstitution inflammatory syndrome (IRIS). Methods: The TCR repertoire in blood and CSF was assessed by complementarity determining region 3 spectratyping in 59 patients with MS treated with natalizumab for at least 18 months, 5 cases of natalizumab-associated PML, 17 age- and sex-matched patients with MS not treated with natalizumab, and 12 healthy controls. Results: Patients with MS presented with peripheral TCR repertoire expansions in blood, which appeared less prominent during therapy with natalizumab. TCR repertoire restrictions observed in CSF were most pronounced in patients with MS treated with natalizumab. In patients who developed PML with longitudinal samples available, new identical TCR receptor length expansions in blood and CSF were observed following plasma exchange, and preceded the development of IRIS. Conclusions: Profound TCR repertoire restrictions in CSF of patients treated with natalizumab reflect an altered immune surveillance of the CNS, which may contribute to an increased risk of developing PML. Natalizumab seems to prompt an impaired or delayed peripheral expansion of antigen-specific T cells, whereas increased reconstitution of peripheral T-cell expansion following plasma exchange may trigger PML-IRIS. Our data suggest that treatment with natalizumab results in broader changes in the T-cell immune repertoire beyond lymphocyte migration.

Background: Neutralizing antibodies (NAbs) to interferon β (IFNβ) products that develop during treatment are associated with a loss of clinical efficacy. Objectives: The aim of this study was to investigate the influence of smoking habits on the risk of developing NAbs to IFNβ, in the treatment of multiple sclerosis (MS). Methods: This report is based on 695 MS patients treated with IFNβ-1a, included in two Swedish case-control studies that collected information on smoking habits. Using logistic regression, the development of NAbs to IFNβ-1a among current smokers was compared with that of non-smokers, by calculating the odds ratio (OR) with a 95% confidence interval (CI). Results: Current smokers showed an increased risk of developing NAbs to IFNβ-1a, compared with non-smokers (OR 1.9; 95% CI 1.3–2.8; p = 0.002). There were no gender differences. We observed no association between past smoking and the risk of developing NAbs to IFNβ-1a. Conclusions: The finding that current smokers have an increased risk of developing NAbs to IFNβ-1a has implications, both for the practical care and the treatment of MS; it also provides an interesting perspective of the lungs as an immune-reactive organ, reacting upon irritation.

BACKGROUND: Human herpesvirus 6 (HHV-6) is an important cause of fulminant or acute viral myocarditis. However, insufficiency of standard antiviral treatment against HHV-6 is an emerging problem. OBJECTIVES: To describe the case of child with HHV-6 myocarditis who was treated by unloading with a left ventricular assist device and Artesunate. STUDY DESIGN: Case report supported by histological and viral diagnoses via a combination of histology/immunohistochemistry and polymerase chain reaction techniques performed on cardiac tissues before and after treatment. RESULTS: Following therapeutic intervention, the clinical status and heart function improved. Endomyocardial biopsies revealed decreased levels of HHV-6B DNA in the myocardium and the disappearance of histological findings in support of lymphocytic myocarditis. Left ventricular assist device could be explanted. No adverse effects of Artesunate were noted. CONCLUSIONS: In addition to existing heart failure treatments, Artesunate can be considered as an effective candidate for clinical use in cases of HHV-6B associated myocarditis. (Less)

Antibodies; Interferon beta; Multiple sclerosis Anticossos; Interferó beta; Esclerosi múltiple Anticuerpos; Interferón beta; Esclerosis múltiple Antibodies against biopharmaceuticals (anti-drug antibodies, ADA) have been a well-integrated part of the clinical care of multiple sclerosis (MS) in several European countries. ADA data generated in Europe during the more than 10 years of ADA monitoring in MS patients treated with interferon beta (IFNβ) and natalizumab have been pooled and characterized through collaboration within a European consortium. The aim of this study was to report on the clinical practice of ADA testing in Europe, considering the number of ADA tests performed and type of ADA assays used, and to determine the frequency of ADA testing against the different drug preparations in different countries. A common database platform (tranSMART) for querying, analyzing and storing retrospective data of MS cohorts was set up to harmonize the data and compare results of ADA tests between different countries. Retrospective data from six countries (Sweden, Austria, Spain, Switzerland, Germany and Denmark) on 20,695 patients and on 42,555 samples were loaded into tranSMART including data points of age, gender, treatment, samples, and ADA results. The previously observed immunogenic difference among the four IFNβ preparations was confirmed in this large dataset. Decreased usage of the more immunogenic preparations IFNβ-1a subcutaneous (s.c.) and IFNβ-1b s.c. in favor of the least immunogenic preparation IFNβ-1a intramuscular (i.m.) was observed. The median time from treatment start to first ADA test correlated with time to first positive test. Shorter times were observed for IFNβ-1b-Extavia s.c. (0.99 and 0.94 years) and natalizumab (0.25 and 0.23 years), which were introduced on the market when ADA testing was already available, as compared to IFNβ-1a i.m. (1.41 and 2.27 years), IFNβ-1b-Betaferon s.c. (2.51 and 1.96 years) and IFNβ-1a s.c. (2.11 and 2.09 years) which were available years before routine testing began. A higher rate of anti-IFNβ ADA was observed in test samples taken from older patients. Testing for ADA varies between different European countries and is highly dependent on the policy within each country. For drugs where routine monitoring of ADA is not in place, there is a risk that some patients remain on treatment for several years despite ADA positivity. For drugs where a strategy of ADA testing is introduced with the release of the drug, there is a reduced risk of having ADA positive patients and thus of less efficient treatment. This indicates that potential savings in health cost might be achieved by routine analysis of ADA.

Background: A small proportion of multiple sclerosis (MS) patients treated with natalizumab develop anti-drug antibodies. Objective: The objective of this paper is to characterize the anti-natalizumab antibody response and to investigate differences between persistently and transiently antibody-positive patients. Methods: Screening for anti-natalizumab antibodies was performed using a standardized bridging ELISA. Antibody-positive samples were further analyzed for IgM and IgG1–4 antibodies using ELISA and ImmunoCAP®. Results: Anti-natalizumab antibodies developed in 57 of 1379 (4.1%) treated patients after a median treatment duration of three months. Of the positive patients, 20 (35%) patients reverted to negative, 19 (33%) patients were confirmed persistently positive and 18 (32%) patients were unconfirmed positive. Significantly higher anti-natalizumab antibody levels were detected in persistently compared to transiently positive patients. A cutoff value predicting persistence of antibodies could be determined with a sensitivity of 0.84 and a specificity of 0.80. IgM and IgG4 antibody levels were significantly higher in persistently compared to transiently positive patients, and IgG1, IgG2 and IgG4 increased significantly over time. Conclusions: The level of total anti-natalizumab antibodies in a first positive sample can be used to predict patients at risk for persisting antibody positivity. However, neither IgM nor IgG1–4 antibodies could be used to discriminate between transiently and persistently positive patients.

Background: Neutralizing antibodies (NAbs) against interferon beta (IFNβ) lead to loss of treatment efficacy in multiple sclerosis patients. The seroprevalence of NAbs in multiple sclerosis patients treated with IFNβ during 2003–2004 was 32% in a cross-sectional analysis of routine data. Objectives: The aim of this study was to investigate whether the seroprevalence of NAbs, the levels of NAb titres and the IFNβ preparations used for treatment of multiple sclerosis patients had changed in 2009–2010. Methods: This study included 1296 patients, analysed for NAbs with the myxovirus resistance protein A gene expression assay in 2009–2010. Results: The seroprevalence of NAbs had decreased to 19% in 2009–2010, which is significantly lower compared with the previous study in 2003–2004 ( pConclusions: NAb seroprevalence has decreased since NAb monitoring became clinical practice in 2003, especially for patients with high NAb titres. This might be due to the stricter monitoring of NAb titres that prompt NAb positive patients to stop treatment, to preferential use of less immunogenic drugs and to alteration of drug formulations.

Background: In the clinical trials about 9% of natalizumab treated multiple sclerosis (MS) patients generated anti-natalizumab antibodies, of which 6% were persistent and 3% transient. The occurrence of antibodies reduced serum levels of natalizumab, decreased bio-efficacy, and abrogated the therapeutic efficacy. Objective: The objective was to assess the frequency of anti-natalizumab antibodies in an unselected cohort of patients from four different countries. Methods: We measured anti-natalizumab antibodies in a large cohort of 4881 unselected patients from four MS centres that systematically measured antibodies in patients treated with natalizumab. We applied the same ELISA assay developed by Biogen Idec and used in the pivotal trials of natalizumab. Results: Antibodies occurred in 4.5% (95% confidence interval, CI: 4.0–5.1%) of the patients, and were persistent in 3.5% (95% CI: 3.0–4.0%) and transient in 1.0% (95% CI: 0.7–1.3%) of the patients. The frequencies of permanently antibody positive patients did not show statistically significant differences between the four centres, whereas the frequencies of transiently antibody positive patients showed some variations. Conclusion: The frequencies of antibodies appeared to be of the same magnitude in the four centres, but might be less than in the pivotal studies of natalizumab.

Background: A post marketing surveillance study was conducted to evaluate safety and efficacy of natalizumab in Swedish multiple sclerosis (MS) patients since its introduction in August 2006 until March 2010. Methods: Patients were registered in the web-based Swedish MS-registry at 40 locations and evaluated every 6 months. Adverse events and clinical outcomes were recorded. Results: One thousand one hundred and fifty-two patients were included (71.4% female) and 149 patients stopped treatment; the main reason was planned pregnancy. Anti-natalizumab antibodies were found in 4.5% (52 patients) of which 1.6% displayed persistent antibodies. Serious adverse events were rare, but included three cases with progressive multifocal leukoencephalopathy (PML). There were seven fatal cases, probably unrelated to the natalizumab treatment. For relapsing–remitting MS patients ( n = 901), mean Expanded Disability Status Scale (EDSS, −10.7%), Multiple Sclerosis Severity Scale (MSSS, −20.4%), Multiple Sclerosis Impact Scale (MSIS-29, physical −9.9%, psychological −13.3%) and Symbol Digit Modalities Test (SDMT, +10.7%) all showed significant improvements during 24 months of treatment with natalizumab. The Swedish web-based MS quality registry proved to function as a platform for post-marketing MS drug surveillance, providing long-term data regarding drug effects and adverse events beyond clinical trials. Conclusions: Our results indicate that natalizumab is generally well tolerated and has sustained efficacy for patients with active MS, though the risk of PML is still an important concern.

Multiple sclerosis patients without cerebrospinal fluid oligoclonal IgG bands have been proposed to constitute an immunogenetically distinct subgroup of multiple sclerosis that may also differ in terms of prognosis. A proportion of patients with multiple sclerosis receiving IFNβ develop neutralizing antibodies, which interfere with treatment efficacy. Evidence suggests that the likelihood of developing neutralizing antibodies is partly genetically determined. Here, we hypothesized that absence of oligoclonal IgG bands reflects a property of B-cell responses in oligoclonal IgG band-negative patients characterized by a lessened propensity to develop neutralizing antibodies. We aimed to compare the development of neutralizing antibodies against IFNβ between oligoclonal IgG band-negative and oligoclonal IgG band-positive multiple sclerosis patients. Treatment, oligoclonal IgG band and neutralizing antibody information was obtained for 2219 patients from the Swedish multiple sclerosis registry and the Swedish neutralizing antibody registry. Additional data on genotype was available for 532 patients. A correlation was found between oligoclonal IgG band negativity and neutralizing antibody negativity ( p = 0.02). This difference was confined to neutralizing antibodies against IFNβ-1a, since oligoclonal IgG band-negative patients were, to a lesser extent, neutralizing antibody positive compared with oligoclonal IgG band-positive patients if treated with IFNβ-1a (12% vs. 23%; p = 0.005). No difference was observed for IFNβ-1b-treated patients (44% vs. 46%). We propose that oligoclonal IgG band-negative patients differ immunologically from oligoclonal IgG band-positive patients, potentially influenced by distinct HLA-DRB1 alleles.

Immunogenicity of biopharmaceutical products in multiple sclerosis is a frequent side effect which has a multifactorial etiology. Here we study associations between anti-drug antibody (ADA) occurrence and demographic and clinical factors. Retrospective data from routine ADA test laboratories in Sweden, Denmark, Austria and Germany (Dusseldorf group) and from one research study in Germany (Munich group) were gathered to build a collaborative multi-cohort dataset within the framework of the ABIRISK project. A subset of 5638 interferon-beta (IFNβ)-treated and 3440 natalizumab-treated patients having data on at least the first two years of treatment were eligible for interval-censored time-to-event analysis. In multivariate Cox regression, IFNβ-1a subcutaneous and IFNβ-1b subcutaneous treated patients were at higher risk of ADA occurrence compared to IFNβ-1a intramuscular-treated patients (pooled HR = 6.4, 95% CI 4.9-8.4 and pooled HR = 8.7, 95% CI 6.6-11.4 respectively). Patients older than 50 years at start of IFNβ therapy developed ADA more frequently than adult patients younger than 30 (pooled HR = 1.8, 95% CI 1.4-2.3). Men developed ADA more frequently than women (pooled HR = 1.3, 95% CI 1.1-1.6). Interestingly we observed that in Sweden and Germany, patients who started IFNβ in April were at higher risk of developing ADA (HR = 1.6, 95% CI 1.1-2.4 and HR = 2.4, 95% CI 1.5-3.9 respectively). This result is not confirmed in the other cohorts and warrants further investigations. Concerning natalizumab, patients older than 45 years had a higher ADA rate (pooled HR = 1.4, 95% CI 1.0-1.8) and women developed ADA more frequently than men (pooled HR = 1.4, 95% CI 1.0-2.0). We confirmed previously reported differences in immunogenicity of the different types of IFNβ. Differences in ADA occurrence by sex and age are reported here for the first time. These findings should be further investigated taking into account other exposures and biomarkers.

Development of neutralizing antibodies (NAbs) reduces the clinical efficacy of interferon beta (IFNβ) treatment in multiple sclerosis (MS) patients. The aim of this study was to evaluate NAb seroprevalence (frequency of patients with NAbs) and immunogenicity (titer levels) of IFNβ preparations in a clinical setting. We analysed 1115 consecutive MS patients, treated with one of the three available IFNβ preparations, for an average of 40 months (1 – 120 months), for the presence of NAbs with the MxA protein induction assay. Overall, 32% of patients were positive for NAbs with neutralizing titers above 10. The frequency of NAbs, ie, the seroprevalence, was 13% in Avonex-treated patients, 43% for Betaferon, 39% for Rebif22 and 30% for Rebif44. In addition, the potential to induce high titer levels, ie, the immunogenicity, was observed to differ between preparations. Avonex, showing the lowest seroprevalence, also showed low immunogenicity and typically induced low titers. Betaferon, showing the highest seroprevalence when inducing NAbs, induced lower titers compared to Rebif22 and Rebif44. Treatment duration over five years only marginally correlated with decreased seroprevalence and titer levels. In conclusion, NAbs to IFNβ are common in a clinical setting and the IFNβ preparations differ not only in NAb seroprevalence, but also in immunogenicity. Multiple Sclerosis 2007; 13: 208–214. http://msj.sagepub.com

When using relative gene expression for quantification of RNA it is crucial that the reference genes used for normalization do not change with the experimental condition. We aimed at investigating the expressional stability of commonly used reference genes during Human herpesvirus 6B (HHV-6B) infection. Expression of eight commonly used reference genes were investigated with quantitative PCR in a T-cell line infected with HHV-6B. The stability of genes was investigated using the 2(-ΔΔCT) method and the algorithms BestKeeper, GeNorm and NormFinder. Our results indicate that peptidylprolyl isomerase A (PPIA) is the most stably expressed gene while TATA box binding protein (TBP) is the least stably expressed gene during HHV-6B infection. In a confirmatory experiment, TBP was demonstrated to be dose and time dependently upregulated by HHV-6B. The stability of PPIA is in line with other studies investigating different herpesvirus infections whereas the finding that HHV-6B significantly upregulates TBP is novel and most likely specific to HHV-6B.

The ultrastructural replication cycle of human herpesvirus 6A and 6B, both T-lymphotropic viruses, with tropism for the central nervous system, was compared by electron microscopy in the same cells, that is, in the T-lymphoblastoid cell line SupT-1 and in human astrocytes. Both HHV-6A and HHV-6B replicated efficiently in SupT-1 and formed viral particles. The tegument is the least characterized structure of the herpesviral particle and both variants were able to form intranuclear membrane compartments called tegusomes in SupT-1 where tegumentation occurred. Also, tegumentation occurred in HHV-6A infected cells in the nucleoplasm without the presence of a tegusome. This suggests that there is more than one possible route of tegumentation. Differences in the replication cycles between HHV-6A and HHV-6B were also observed in the cytoplasm. One such difference was that prominent annulate lamellae were only found in the cytoplasm of HHV-6A infected cells. In astrocytes a successful formation of viral particles was only seen with the HHV-6A variant. The HHV-6A virus life cycle in astrocytes resembled the life cycle in the T-cell line SupT-1, except that no annulate lamellae were found. Complete viral particles were found extracellularly around the astrocytes and the supernatant of infected astrocytes were able to re-infect SupT-1 cells. This suggests that HHV-6A infection in astrocytes can generate complete, viable, and infectious viral particles. The HHV-6 variants behave differently in the same type of cells and have different tropisms for astrocytes, supporting the notion that the variants might induce different diseases.

Human herpesvirus 6 (HHV-6) is a ubiquitous beta -herpesvirus associated with a number of clinical disorders. Two closely but biologically distinct variants have been described. HHV-6 variant B causes the common childhood disease exhanthem subitum, and although the pathologic characteristics for HHV-6 variant A are less well defined, HHV-6A has been suggested to be more neurotropic. We studied the effect of both HHV-6 variants in an oligodendrocyte cell line (MO3.13). Infection of M03.13 was monitored by cytopathic effect (CPE), quantitative TaqMan PCR for viral DNA in cells and supernatant, reverse transcriptase-polymerase chain reaction (RT-PCR) to detect viral RNA, and indirect immunofluorescence (IFA) to detect viral protein expression. HHV-6A infection induced significantly more CPE than infection with HHV-6B. HHV-6B induced an abortive infection associated with a decrease of the initial viral DNA load over time, early RNA expression, and no expression of viral antigen. In contrast, infection with HHV-6A DNA persisted in cells for at least 62 days. During the acute phase of infection with HHV-6A, intracellular and extracellular viral load increased and cells expressed the viral protein IE-2 and gp116/54/64. No HHV-6A RNA or protein was expressed after 30 days post infection, suggesting that HHV-6A formed a latent infection. These studies provide in vitro support to the hypothesis that HHV-6 can actively infect oligodendrocytes. Our results suggest that HHV-6A and HHV-6B have different tropism in MO3.13 cells and that an initially active HHV-6A infection can develop latency. Differences between HHV-6A and -6B infection in different neural cell types may be associated with different neurological diseases.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) with supposedly autoimmune features known to be associated with a specific HLA DR-DQ haplotype (DR15, DQ6, or HLDRB1*1501,DRB5*0101,DQA1*0102,DQB1*0602). We have previously reported that the associated haplotype extends to HLA-B and described an independent association with HLA-A alleles in MS. Owing to a complex situation with extensive linkage disequilibria, it is still unclear whether classical HLA genes are responsible or whether associations may be due to other genes in this region. Here, we analyzed an association in MS with the NOTCH4 and TNFalpha (tumor necrosis factor-alpha) genes, located between the HLA-DRB1 gene and the HLA-A gene. For NOTCH4, located 0.4 Mb telomeric to HLA-DRB1, an SNP at position -25 and a trinucleotide repeat were investigated in 181 MS patients, and 180 controls also typed P = 0.027 for HLA-DRB and HLA-A. A modest association was observed (OR = 3.44) with the C-25 allele. However, two-locus analysis revealed that this association was secondary to the classical association with HLA-DRB1. For TNF, located 0.7 Mb telomeric of NOTCH4, SNPs at positions -308 and -238 were studied in the same dataset. We found no association between these TNFalpha gene polymorphisms and MS in this dataset, although there was linkage disequilibrium (LD) between DRB1 and TNF and between HLA-A and TNF. We conclude that alleles of the NOTCH4 and TNFalpha genes are unlikely to be of importance for the susceptibility to MS, although specific alleles of these genes are often carried on the same haplotype as DR15, DQ6.

The human herpesvirus-6 (HHV-6) is a ubiquitous β-herpesvirus,1,2 which infects a wide spectrum of cell types including glial cells.3–5 The virus is acquired early in childhood and is the causative agent of roseola infantum (exanthem subitum), a benign and otherwise self-limiting disease.6 Primary infection may result in seizures and severe neurologic complications such as meningitis or meningoenceph-alitis.7 As with all herpesviruses, HHV-6 can establish lifelong latent infection,8 and reactivation can occur in immunosuppressed patients such as bone marrow transplant recipients.9 Two HHV-6 variants have been characterized with respect to their antigenic and genomic composition.10 HHV-6B is primarily associated with most symptomatic infections during infancy,11 whereas HHV-6A has been suggested to be more neurotropic and is associated with viral persistence and reactivation in the CNS.12 HHV-6 variant A has also been detected more frequently in multiple sclerosis patient samples compared with variant B.13 However, HHV-6B has also been shown to have neurotropic potential4,14 and has recently been associated with limbic encephalitis.15 Although the role of HHV-6 in human CNS disease remains to be fully defined, a number of studies have suggested that the CNS can be a site for persistent HHV-6 infection.16,17 Autopsy material from a wide variety of normal18 and diseased19,20 brains has documented HHV-6 infection in the CNS, although its role as a causative agent is unclear. Cell types reported to be infected include oligodendrocytes, astrocytes, and possibly neurons.21,22 In limbic encephalitis, HHV-6 DNA was detected in the CSF, and astrocytes were shown to be the most represented cell type harboring HHV-6B in the hippocampus.15 This localization is consistent with the report of HHV-6 hippocampal encephalitis after bone marrow transplantation23 and of hippocampal injury in patients with prolonged focal febrile seizures,24,25 a frequent complication of a primary HHV-6 infection. In an attempt to characterize further the extent and distribution of HHV-6 in human glial cells, brain tissues from surgical specimens were processed to establish cultures of primary astrocytes and oligodendrocytes to be used for in vitro virologic examinations. Brain resections are a therapy for pharmacologically untreatable seizures,26 and specimens from patients with mesial temporal lobe epilepsy (MTLE) and neocortical epilepsy (NE) were collected for analysis.

Background: Interleukin-7 (IL-7) is a non-redundant cytokine for T-cell development and survival. The IL-7 signaling pathway has been genetically and functionally associated with several autoimmune diseases including multiple sclerosis (MS). Objective: The objective of this paper is to elucidate the effect of the widely used immunomodulatory MS therapy interferon beta (IFNβ) on IL-7 homeostasis. Methods: Swedish MS patients were screened for IL-7 concentration in serum and blood cell counts. IL-7 receptor alpha chain (IL-7Rα) expression was determined by semi-quantitative real-time polymerase chain reaction (PCR) and flow cytometry. Results: IFNβ treatment led to significantly increased serum IL-7 levels (mean: 17 pg/ml) compared with healthy controls (mean: 7.6 pg/ml) and natalizumab-treated patients (mean: 5.3 pg/ml). In vitro and in vivo, peripheral blood leukocytes showed decreased IL-7Rα expression and IL-7 consumption upon IFNβ exposure, suggesting that their IL-7 responsiveness is impaired during treatment. Conclusions: MS patients undergoing IFNβ treatment have increased serum IL-7 levels and decreased IL-7 consumption. Given IL-7’s important role in T-cell immunity, this relationship may be highly relevant for IFNβ’s treatment efficacy.

The advent of biopharmaceuticals (BPs) has led to significant improvements in the treatment of many chronic inflammatory diseases, and the number of BPs on the market and of diseases treated reflects their success. However, repetitive parenteral administration and intrinsic immunogenic properties of the drug can elicit an immune response, leading to production of anti-drug antibodies (ADA). This is a major limitation of the use of BPs and has to be taken into consideration in clinical practice and during drug development. With increasing knowledge about the immunogenicity of BPs and regular ADA testing in patients, we ensure optimized long-term treatment for the individual and thus optimal use of health care resources. This field has already been extensively investigated in the treatment of multiple sclerosis with IFN-β, but there is a clear need for consensus from academia, health care providers and the BP industry in managing ADA across all BPs and diseases.

Membrane cofactor protein (CD46) is a member of a family of glycoproteins that are regulators of complement and prevent activation of complement on autologous cells. Recently, CD46 has been identified as the cellular receptor for human herpesvirus Type 6 (HHV-6). Elevated levels of soluble CD46 have been described in several autoimmune disorders, and may be implicated in the pathogenesis of these diseases. As several reports have supported an association of HHV-6 and multiple sclerosis, it was of interest to compare levels of soluble CD46 in the sera of multiple sclerosis patients to that of healthy controls, other neurological disease controls, and other inflammatory disease controls. Using an immunoaffinity column comprised of immobilized monoclonal antibodies to CD46, serum levels of soluble CD46 were found to be significantly elevated in multiple sclerosis patients compared with healthy and other neurological disease controls. Moreover, multiple sclerosis patients who tested positive for HHV-6 DNA in serum had significantly elevated levels of soluble CD46 in their serum compared with those who were negative for HHV-6 DNA. A significant increase in soluble CD46 was also found in the serum of other inflammatory disease controls tested compared to healthy controls. Additionally, a significant correlation was demonstrated between levels of soluble CD46 in the serum and cerebrospinal fluid of multiple sclerosis patients. Collectively, these data suggest that elevated levels of soluble CD46 may contribute to the pathogenesis of inflammatory diseases, including multiple sclerosis.

Interferon beta (IFNβ) is used as a first-line treatment in relapsing-remitting multiple sclerosis (MS). The occurrence of neutralizing antidrug antibodies (NAbs) against IFNβ may reduce treatment response. Therefore, clinical monitoring of NAbs is currently executed using bioassays, but several bioassays are available and it is unclear how well their readouts correlate. We made a comparison between 2 bioassays; myxovirus resistance protein A (MxA) gene expression assay (MGA) and iLite™ anti-Human IFNβ bioassay, to measure IFNβ-specific NAb titers in 44 MS patients. We further studied how NAb titers affected in vivo transcription of IFN-induced genes myxovirus resistant 1 (MX1) and C-X-C motif chemokine 10 (CXCL10), in addition to serum CXCL10 protein levels. There were significant correlations between NAb titer levels measured with MGA and iLite (Spearman r=0.9368). MX1 and CXCL10 gene expression was strongly induced by IFNβ and NAb positivity significantly reduced this expression. A NAb titer of 150 TRU/mL was observed to be a biological cut-point applicable to both assays, since MX1 and CXCL10 expression was greatly reduced or blocked in patients above this titer level. In conclusion, NAb titers measured with the MGA and iLite bioassays are comparable, but the threshold for positivity in both assays does not correspond to the biologically functional cut-point.