Anti-rituximab antibodies demonstrate neutralizing capacity, associate with lower circulating drug levels and earlier relapse in lupus.

OBJECTIVES: High rates of anti-drug antibodies (ADA) to rituximab have been demonstrated in patients undergoing treatment for SLE. However, little is known with regard to their long-term dynamics, impact on drug kinetics and subsequent implications for treatment response. In this study, we aimed to evaluate ADA persistence over time, impact on circulating drug levels, assess clinical outcomes and whether they are capable of neutralizing rituximab. METHODS: Patients with SLE undergoing treatment with rituximab were recruited to this study (n = 35). Serum samples were collected across a follow-up period of 36 months following treatment (n = 114). Clinical and laboratory data were collected pre-treatment and throughout follow-up. ADA were detected via electrochemiluminescent immunoassays. A complement dependent cytotoxicity assay was used to determine neutralizing capacity of ADA in a sub-cohort of positive samples (n = 38). RESULTS: ADA persisted over the 36-month study period in 64.3% of patients undergoing treatment and titres peaked earlier and remained higher in those who had previously been treated with rituximab when compared with than those who were previously treatment naive. ADA-positive samples had a significantly lower median drug level until six months post rituximab infusion (P = 0.0018). Patients with persistent ADA positivity showed a significant early improvement in disease activity followed by increased rates of relapse. In vitro analysis confirmed the neutralizing capacity of ADA to rituximab. CONCLUSIONS: ADA to rituximab were common and persisted over the 36-month period of this study. They associated with earlier drug elimination, an increased rate of relapse and demonstrated neutralizing capacity in vitro.

Short report: Real-life analysis of the occurrence of persistent, transient, and fluctuating positive titres of neutralizing anti-drug antibodies in multiple sclerosis patients treated with interferon beta.

Interferon beta (IFNß) 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 IFNß, 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 IFNß-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.

Rituximab in Systemic Lupus Erythematosus: Transient Effects on Autoimmunity Associated Lymphocyte Phenotypes and Implications for Immunogenicity.

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 (TFH, CD4+PD-1+CXCR5+) and of peripheral helper T cells (TPH, CD4+PD-1+CXCR5-) did not change after RTX. A sub-cluster of PD-1highCD4+ T cells showed a significant decrease at later follow-up compared to early follow-up (p=0.0039). It is well appreciated that RTX transiently influences B cells. Here, we extend these observations to cell phenotypes which are believed to directly contribute to autoimmunity in SLE. We show early transient effects of RTX on ABC-like memory B cells, later effects on PD-1high CD4+ cells, and possible implications for RTX immunogenicity. Further insight in such effects and their monitoring may be of clinical relevance.

Standardized mortality ratios in multiple sclerosis: Systematic review with meta-analysis.

OBJECTIVE: To perform a meta-analysis of all-cause, cause-specific and gender-specific standardized mortality ratio and crude mortality rate for people with multiple sclerosis. We also examined the temporal trends in this data. METHODS: Medline, Cochrane Library and Scopus were searched. Keywords were "multiple sclerosis" and "standardized mortality ratio" or "Standardized Mortality Ratio". We included longitudinal studies with available data on the number of deaths, follow-up period, person years and reports of standardized mortality ratio (SMR). Crude mortality ratio (CMR) was calculated and SMR was extracted. CMRs and log-SMR were pooled by the method of inverse variance. Meta-regression models were used to investigate temporal trends. RESULTS: Fifty-seven articles were screened. Fifteen studies were included covering a period 1949-2013 (160,000 patients; 21,225 deaths). The all-cause SMR for people with MS was 2.61 (95% CI 2.58 to 2.65). For men this was 2.47 (95% CI 2.42 to 2.52) and for women 2.57 (95% CI 2.53 to 2.61). The CMR was 13.45/1000 person years. Cause-specific SMR was 1.74 (1.67 to 1.81) for CVD, 4.70 (4.45 to 4.87) for respiratory disease and infection, 1.81 (1.64 to 2.0) for accident and suicide and 0.99 (0.93 to 1.06) for cancer. Meta-regression analysis of the SMR compared to midpoint follow-up year revealed no relationship (co-efficient 0.001, p = .98). CONCLUSIONS: People with multiple sclerosis (MS) have reduced overall survival and increased risk of death from cardiovascular, respiratory and infectious disease as well as accidents and suicide. This does not appear to have changed over the last 65 years.

First exposure to rituximab is associated to high rate of anti-drug antibodies in systemic lupus erythematosus but not in ANCA-associated vasculitis.

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 < 0.0001), ADA-positive individuals still had higher SLEDAI-2 K (6.0 (4.0-9.0) vs 4.0 (2.0-6.0), 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.

False Positive Results in SARS-CoV-2 Serological Tests for Samples From Patients With Chronic Inflammatory Diseases.

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.

Tennis-specific extension of the International Olympic Committee consensus statement: methods for recording and reporting of epidemiological data on injury and illness in sport 2020.

The IOC has proposed standard methods for recording and reporting of data for injury and illness in sport. The IOC consensus statement authors anticipated that sport-specific statements would provide further recommendations. This statement is the tennis-specific extension of the partner IOC statement. The International Tennis Federation Sport Science and Medicine Committee, in collaboration with selected external experts, met in June 2019 to consider athlete health monitoring issues specific to tennis. Once the IOC consensus statement was finalised, the tennis-specific consensus was drafted and agreed on by the members over three iterations. Compared with the IOC consensus statement, the tennis consensus contains tennis-specific information on injury mechanism, mode of onset, injury classification, injury duration, capturing and reporting exposure, reporting risk and study population. Our recommendations apply to able-bodied as well as wheelchair tennis players. Where applicable, specific recommendations are made for wheelchair tennis.

Long-Term Consequences of High Titer Neutralizing Antibodies to Interferon-ß in Multiple Sclerosis.

Background: Neutralizing anti-drug antibodies (NAbs) to interferon beta (IFNß) develop in up to 47% of multiple sclerosis (MS) treated patients inhibiting treatment effect of IFNß. However, the long-term effect of NAbs remain unknown. Objective: To investigate the long-term consequences of high titer NAbs to IFNß on disease activity and progression in MS patients. Methods: An observational study including data from all IFNß 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 IFNß 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 IFNß are associated with higher disease activity, persisting even after IFNß discontinuation or switch. These results support use of highly efficient treatment earlier in patients with active disease, to avoid these complications.

The role of herpesvirus 6A and 6B in multiple sclerosis and epilepsy.

Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) are two closely related viruses that can infect cells of the central nervous system (CNS). The similarities between these viruses have made it difficult to separate them on serological level. The broad term HHV-6 remains when referring to studies where the two species were not distinguished, and as such, the seroprevalence is over 90% in the adult population. HHV-6B has been detected in up to 100% of infants with the primary infection roseola infantum, but less is known about the primary infection of HHV-6A. Both viruses are neurotropic and have capacity to establish lifelong latency in cells of the central nervous system, with potential to reactivate and cause complications later in life. HHV-6A infection has been associated with an increased risk of multiple sclerosis (MS), whereas HHV-6B is indicated to be involved in pathogenesis of epilepsy. These two associations show how neurological diseases might be caused by viral infections, but as suggested here, through completely different molecular mechanisms, in an autoimmune disease, such as MS, by triggering an overreaction of the immune system and in epilepsy by hampering internal cellular functions when the immune system fails to eliminate the virus. Understanding the viral mechanisms of primary infection and reactivation and their spectrum of associated symptoms will aid our ability to diagnose, treat and prevent these severe and chronic diseases. This review explores the role of HHV-6A and HHV-6B specifically in MS and epilepsy, the evidence to date and the future directions of this field.

Drug Tolerant Anti-drug Antibody Assay for Infliximab Treatment in Clinical Practice Identifies Positive Cases Earlier.

A subgroup of patients treated with infliximab lose response to the treatment and one reason for this is the development of anti-drug antibodies (ADA). If used optimally, measuring drug and ADA level could lead to a more personalized and efficient treatment regime, and enable identification of ADA-positive patients before the underlying disease flares or allergic reactions occur. With the use of a drug-tolerant ADA assay which can detect ADA irrespective of drug levels in the sample, we determined the impact of ADA on treatment failure to infliximab. The aims of this study were to estimate the real-life optimal serum infliximab (sIFX) level and set a clinical threshold value for a drug-tolerant ADA assay. Trough levels of sIFX were measured with ELISA. Free ADA was measured with two drug-sensitive methods (ELISA and a bioassay) and one drug-tolerant method (PandA). Two real-life cohorts treated with infliximab were included; a cross-sectional cohort including patients with inflammatory rheumatic diseases (n = 270) and a prospective cohort of rheumatoid arthritis (RA) patients (n = 73) followed for 1 year. Normal range of sIFX was estimated from the prospective cohort and an arbitrary optimal drug level was set to be between 1 and 6 µg/mL. Using this range, optimal sIFX was found in only 60% (163/270) of the patients in the cross-sectional cohort. These patients had significantly better treatment response than those with a drug level under 1 µg/mL, who had an ADA frequency of 34% (19/56) using the drug-tolerant method. In the prospective cohort, the drug-tolerant assay could identify 34% (53/155 samples) as ADA positive in samples with sIFX level >0.2 µg/mL. ADA were seldom detected in patients with >1 µg/mL sIFX, with three interesting exceptions. A clinically relevant ADA threshold was determined to be >3 RECL as measured with the drug-tolerant assay. In a real-life setting, there was a substantial number of patients with suboptimal drug levels and a proportion of these had ADA. Both too low and too high drug levels correlated with worse disease, but for different reasons. Adding a drug-tolerant assay enabled detection of ADA earlier and regardless of drug level at time of sampling.

Rituximab in multiple sclerosis: Frequency and clinical relevance of anti-drug antibodies.

BACKGROUND: Rituximab is a chimeric monoclonal anti-CD20 B-cell-depleting antibody increasingly used off-label in multiple sclerosis (MS). The clinical relevance of anti-drug antibodies (ADAs) against rituximab in MS is unknown. OBJECTIVE: To determine frequency of ADA in relation to B-cell counts, allergic reactions and clinical efficacy in a large cohort of MS-treated patients. METHODS: Cross-sectional study with collection of serum samples from 339 MS patients immediately before a scheduled rituximab infusion. ADAs were detected using an in-house-validated electrochemiluminescent immunoassay and a commercial enzyme-linked immunosorbent assay (ELISA) to compare methods. Data on patient demographics and clinical outcomes were retrieved from the Swedish MS Registry and patient records. RESULTS: ADAs were detected in 37% of relapsing-remitting MS and 26% in progressive forms of MS. Presence of ADAs decreased with increasing number of rituximab infusions. There was a significant association between both presence and titres of ADAs and incomplete B-cell depletion, but not with infusion/adverse reactions or clinical outcomes at the group level. Only five patients terminated rituximab during follow-up, four of which were ADA positive. CONCLUSION: Rituximab treatment is associated with a high degree of ADAs, which correlates with efficacy of B-cell depletion; however, the clinical relevance of ADAs remains uncertain.

Human Herpesvirus 6B Induces Hypomethylation on Chromosome 17p13.3, Correlating with Increased Gene Expression and Virus Integration.

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 <1 Mb from chromosomal ends and were all hypomethylated in virus-infected cells. This was most evident at chromosome 17p13.3, where HHV-6B had induced CpG hypomethylation after 2 days of infection, possibly through TET2, which was found to be upregulated by the virus. In addition, virus-induced cytosine hydroxymethylation was observed. Genes located in the hypomethylated region at 17p13.3 showed significantly upregulated expression in HHV-6B-infected cells. A temporal experiment revealed HHV-6B integration in Molt-3 cell DNA 3 days after infection. The telomere at 17p has repeatedly been described as an integration site for HHV-6B, and we show for the first time that HHV-6B induces hypomethylation in this region during acute infection, which may play a role in the integration process, possibly by making the DNA more accessible.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.

Investigation of reference gene expression during human herpesvirus 6B infection indicates peptidylprolyl isomerase A as a stable reference gene and TATA box binding protein as a gene up-regulated by this virus.

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.