Risk of COVID-19 in people with multiple sclerosis who are seronegative following vaccination.

BACKGROUND: People with multiple sclerosis (pwMS) treated with certain disease-modifying therapies (DMTs) have attenuated IgG response following COVID-19 vaccination; however, the clinical consequences remain unclear. OBJECTIVE: To report COVID-19 rates in pwMS according to vaccine serology. METHODS: PwMS with available (1) serology 2-12 weeks following COVID-19 vaccine 2 and/or vaccine 3 and (2) clinical data on COVID-19 infection/hospitalisation were included. Logistic regression was performed to examine whether seroconversion following vaccination predicted risk of subsequent COVID-19 infection after adjusting for potential confounders. Rates of severe COVID-19 (requiring hospitalisation) were also calculated. RESULTS: A total of 647 pwMS were included (mean age 48 years, 500 (77%) female, median Expanded Disability Status Scale (EDSS) 3.5% and 524 (81%) exposed to DMT at the time of vaccine 1). Overall, 472 out of 588 (73%) were seropositive after vaccines 1 and 2 and 222 out of 305 (73%) after vaccine 3. Seronegative status after vaccine 2 was associated with significantly higher odds of subsequent COVID-19 infection (odds ratio (OR): 2.35, 95% confidence interval (CI): 1.34-4.12, p = 0.0029), whereas seronegative status after vaccine 3 was not (OR: 1.05, 95% CI: 0.57-1.91). Five people (0.8%) experienced severe COVID-19, all of whom were seronegative after most recent vaccination. CONCLUSION: Attenuated humoral response to initial COVID-19 vaccination predicts increased risk of COVID-19 in pwMS, but overall low rates of severe COVID-19 were seen.

Response to COVID-19 booster vaccinations in seronegative people with multiple sclerosis.

BACKGROUND: People with MS treated with anti-CD20 therapies and fingolimod often have attenuated responses to initial COVID-19 vaccination. However, uncertainties remain about the benefit of a 3rd (booster) COVID-19 vaccine in this group. METHODS: PwMS without a detectable IgG response following COVID-19 vaccines 1&2 were invited to participate. Participants provided a dried blood spot +/- venous blood sample 2-12 weeks following COVID-19 vaccine 3. Humoral and T cell responses to SARS-CoV-2 spike protein and nucleocapsid antigen were measured. RESULTS: Of 81 participants, 79 provided a dried blood spot sample, of whom 38 also provided a whole blood sample; 2 provided only whole blood. Anti-SARS-CoV-2-spike IgG seroconversion post-COVID-19 vaccine 3 occurred in 26/79 (33%) participants; 26/40 (65%) had positive T-cell responses. Overall, 31/40 (78%) demonstrated either humoral or cellular immune response post-COVID-19 vaccine 3. There was no association between laboratory evidence of prior COVID-19 and seroconversion following vaccine 3. CONCLUSIONS: Approximately one third of pwMS who were seronegative after initial COVID-19 vaccination seroconverted after booster (third) vaccination, supporting the use of boosters in this group. Almost 8 out of 10 had a measurable immune response following 3rd COVID-19 vaccine.

COVID-19 Vaccine Response in People with Multiple Sclerosis.

OBJECTIVE: The purpose of this study was to investigate the effect of disease modifying therapies on immune response to severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) vaccines in people with multiple sclerosis (MS). METHODS: Four hundred seventy-three people with MS provided one or more dried blood spot samples. Information about coronavirus disease 2019 (COVID-19) and vaccine history, medical, and drug history were extracted from questionnaires and medical records. Dried blood spots were eluted and tested for antibodies to SARS-CoV-2. Antibody titers were partitioned into tertiles with people on no disease modifying therapy as a reference. We calculated the odds ratio of seroconversion (univariate logistic regression) and compared quantitative vaccine response (Kruskal Wallis) following the SARS-CoV-2 vaccine according to disease modifying therapy. We used regression modeling to explore the effect of vaccine timing, treatment duration, age, vaccine type, and lymphocyte count on vaccine response. RESULTS: Compared to no disease modifying therapy, the use of anti-CD20 monoclonal antibodies (odds ratio = 0.03, 95% confidence interval [CI] = 0.01-0.06, p < 0.001) and fingolimod (odds ratio = 0.04; 95% CI = 0.01-0.12) were associated with lower seroconversion following the SARS-CoV-2 vaccine. All other drugs did not differ significantly from the untreated cohort. Both time since last anti-CD20 treatment and total time on treatment were significantly associated with the response to the vaccination. The vaccine type significantly predicted seroconversion, but not in those on anti-CD20 medications. Preliminary data on cellular T-cell immunity showed 40% of seronegative subjects had measurable anti-SARS-CoV-2 T cell responses. INTERPRETATION: Some disease modifying therapies convey risk of attenuated serological response to SARS-CoV-2 vaccination in people with MS. We provide recommendations for the practical management of this patient group. ANN NEUROL 20219999:n/a-n/a.

CSF-resident CD4+ T-cells display a distinct gene expression profile with relevance to immune surveillance and multiple sclerosis.

The CNS has traditionally been considered an immune privileged site, but is now understood to have a system of immune surveillance, predominantly involving CD4+ T-cells. Identifying functional differences between CNS and blood CD4+ T-cells, therefore, have relevance to CNS immune surveillance as well as to neurological conditions, such as multiple sclerosis, in which CD4+ T-cells play a central role. Here, CD4+ T-cells were purified from CSF and blood from 21 patients with newly diagnosed treatment-naïve multiple sclerosis and 20 individuals with non-inflammatory disorders using fluorescence-activated cell sorting, and their transcriptomes were profiled by RNA sequencing. Paired comparisons between CD4+ T-cells from CSF and blood identified 5156 differentially expressed genes in controls and 4263 differentially expressed in multiple sclerosis patients at false discovery rate <5%. Differential expression analysis of CD4+ T-cells collected from the CSF highlighted genes involved in migration, activation, cholesterol biosynthesis and signalling, including those with known relevance to multiple sclerosis pathogenesis and treatment. Expression of markers of CD4+ T-cell subtypes suggested an increased proportion of Th1 and Th17 cells in CSF. Gene ontology terms significant only in multiple sclerosis were predominantly those involved in cellular proliferation. A two-way comparison of CSF versus blood CD4+ T-cells in multiple sclerosis compared with non-inflammatory disorder controls identified four significant genes at false discovery rate <5% (CYP51A1, LRRD1, YES1 and PASK), further implicating cholesterol biosynthesis and migration mechanisms. Analysis of CSF CD4+ T-cells in an extended cohort of multiple sclerosis cases (total N = 41) compared with non-inflammatory disorder controls (total N = 38) identified 140 differentially expressed genes at false discovery rate < 5%, many of which have known relevance to multiple sclerosis, including XBP1, BHLHE40, CD40LG, DPP4 and ITGB1. This study provides the largest transcriptomic analysis of purified cell subpopulations in CSF to date and has relevance for the understanding of CNS immune surveillance, as well as multiple sclerosis pathogenesis and treatment discovery.

A blood-based metabolomics test to distinguish relapsing-remitting and secondary progressive multiple sclerosis: addressing practical considerations for clinical application.

The transition from relapsing-remitting multiple sclerosis (RRMS) to secondary progressive MS (SPMS) represents a huge clinical challenge. We previously demonstrated that serum metabolomics could distinguish RRMS from SPMS with high diagnostic accuracy. As differing sample-handling protocols can affect the blood metabolite profile, it is vital to understand which factors may influence the accuracy of this metabolomics-based test in a clinical setting. Herein, we aim to further validate the high accuracy of this metabolomics test and to determine if this is maintained in a 'real-life' clinical environment. Blood from 31 RRMS and 28 SPMS patients was subjected to different sample-handling protocols representing variations encountered in clinics. The effect of freeze-thaw cycles (0 or 1) and time to erythrocyte removal (30, 120, or 240 min) on the accuracy of the test was investigated. For test development, samples from the optimised protocol (30 min standing time, 0 freeze-thaw) were used, resulting in high diagnostic accuracy (mean ± SD, 91.0 ± 3.0%). This test remained able to discriminate RRMS and SPMS samples that had experienced additional freeze-thaw, and increased standing times of 120 and 240 min with accuracies ranging from 85.5 to 88.0%, because the top discriminatory metabolite biomarkers from the optimised protocol remained discriminatory between RRMS and SPMS despite these sample-handling variations. In conclusion, while strict sample-handling is essential for the development of metabolomics-based blood tests, the results confirmed that the RRMS vs. SPMS test is resistant to sample-handling variations and can distinguish these two MS stages in the clinics.

Detecting and predicting neutralization of alemtuzumab responses in MS.

OBJECTIVE: To test the hypothesis that antidrug antibodies (ADAs) against alemtuzumab could become relevant after repeated treatments for some individuals, possibly explaining occasional treatment resistance. METHODS: Recombinant alemtuzumab single-chain variable fragment antibody with a dual tandem nanoluciferase reporter linker was made and used to detect binding ADAs. Alemtuzumab immunoglobulin G Alexa Fluor 488 conjugate was used in a competitive binding cell-based assay to detect neutralizing ADAs. The assays were used to retrospectively screen, blinded, banked serum samples from people with MS (n = 32) who had received 3 or more cycles of alemtuzumab. Lymphocyte depletion was measured between baseline and about 1 month postinfusion. RESULTS: The number of individuals showing limited depletion of lymphocytes increased with the number of treatment cycles. Lack of depletion was also a poor prognostic feature for future disease activity. ADA responses were detected in 29/32 (90.6%) individuals. Neutralizing antibodies occurred before the development of limited depletion in 6/7 individuals (18.8% of the whole sample). Preinfusion, ADA levels predicted limited, postinfusion lymphocyte depletion. CONCLUSIONS: Although ADAs to alemtuzumab have been portrayed as being of no clinical significance, alemtuzumab-specific antibodies appear to be clinically relevant for some individuals, although causation remains to be established. Monitoring of lymphocyte depletion and the antidrug response may be of practical value in patients requiring additional cycles of alemtuzumab. ADA detection may help to inform on retreatment or switching to another treatment.

Cerebrospinal fluid complement system biomarkers in demyelinating disease.

BACKGROUND: Multiple sclerosis (MS) can be difficult to differentiate from other demyelinating diseases, notably neuromyelitis optica spectrum disorder (NMOSD). We previously showed that NMOSD is distinguished from MS by plasma complement biomarkers. OBJECTIVE: Here, we measure cerebrospinal fluid (CSF) complement proteins in MS, NMOSD and clinically isolated syndrome (CIS), a neurological episode that may presage MS, to test whether these distinguish NMOSD from MS and CIS. MATERIALS AND METHODS: CSF (53 MS, 17 CIS, 11 NMOSD, 35 controls) was obtained; complement proteins (C4, C3, C5, C9, C1, C1q, Factor B (FB)), regulators (Factor I (FI), Factor H (FH), FH-Related Proteins 1, 2 and 5 (FHR125), C1 Inhibitor (C1INH), Properdin) and activation products (terminal complement complex (TCC), iC3b) were quantified by ELISA and results expressed relative to CSF total protein (µg/mg). RESULTS: Compared to control CSF, (1) levels of C4, C1INH and Properdin were elevated in MS; (2) TCC, iC3b, FI and FHR125 were increased in CIS; and (3) all complement biomarkers except TCC, FHR125, Properdin and C5 were higher in NMOSD CSF. A statistical model comprising six analytes (C3, C9, FB, C1q, FI, Properdin) plus age/gender optimally differentiated MS from NMOSD.

Longitudinal Characterization of Autoantibodies to the Thyrotropin Receptor (TRAb) During Alemtuzumab Therapy: Evidence that TRAb May Precede Thyroid Dysfunction by Many Years.

BACKGROUND: Thyroid autoimmunity, especially Graves' disease or hypothyroidism with positive autoantibodies (TRAb) to the thyrotropin receptor (TSHR), occurs in 30-40% of patients with relapsing multiple sclerosis following treatment with alemtuzumab (ALTZ). ALTZ therapy therefore provides a unique opportunity to study the evolution of TRAb prior to clinical presentation. TRAb can stimulate (TSAb), block (TBAb), or not affect ("neutral") the TSHR function, causing hyperthyroidism, hypothyroidism, or euthyroidism, respectively. METHODS: A longitudinal retrospective analysis was conducted of TRAb bioactivity over a period of nine years in 45 multiple sclerosis patients receiving ALTZ using available stored serum. Of these 45 patients, 31 developed thyroid dysfunction (TD) and 14 remained euthyroid despite being followed for a minimum of five years (NO-TD). The presence of TRAb was evaluated at standardized time points: (i) before ALTZ, (ii) latest time available following ALTZ and before TD onset, and (iii) following ALTZ during/after TD onset. Serum TRAb were detected by published in-house assays (ihTRAb): flow cytometry detecting any TSHR-binding TRAb, and luciferase bioassays detecting TSAb/TBAb bioactivity. Purified immunoglobulin G was used to verify TSAb/TBAb in selected hypothyroid cases. Standard clinical automated measurements of TRAb, antithyroid peroxidase autoantibodies (TPOAb), thyrotropin, free thyroxine, and free triiodothyronine were also collected. RESULTS: Before ALTZ, combined ihTRAb (positive with flow cytometry and/or luciferase bioassay) but not automated TRAb were present in 5/16 (31.2%) TD versus 0/14 (0%) NO-TD (p = 0.017). Detectable ihTRAb preceded TD development in 9/28 (32.1%) and by a median of 1.2 years (range 28 days-7.3 years). Combination testing of ihTRAb and TPOAb at baseline predicted 20% of subsequent cases of hyperthyroidism and 83% of hypothyroidism. CONCLUSIONS: Evidence is presented that TRAb measured with custom-made assays can be detected prior to any change in thyroid function in up to a third of cases of ALTZ-related TD. Furthermore, the presence of ihTRAb prior to ALTZ treatment was strongly predictive of subsequent TD. The findings suggest that a period of affinity maturation of TRAb may precede clinical disease onset in some cases. Combined testing of TPOAb and ihTRAb may increase the ability to predict those who will develop TD following ALTZ.

Complement system biomarkers in epilepsy.

PURPOSE: To explore whether complement dysregulation occurs in a routinely recruited clinical cohort of epilepsy patients, and whether complement biomarkers have potential to be used as markers of disease severity and seizure control. METHODS: Plasma samples from 157 epilepsy cases (106 with focal seizures, 46 generalised seizures, 5 unclassified) and 54 controls were analysed. Concentrations of 10 complement analytes (C1q, C3, C4, factor B [FB], terminal complement complex [TCC], iC3b, factor H [FH], Clusterin [Clu], Properdin, C1 Inhibitor [C1Inh] plus C-reactive protein [CRP]) were measured using enzyme linked immunosorbent assay (ELISA). Univariate and multivariate statistical analysis were used to test whether combinations of complement analytes were predictive of epilepsy diagnoses and seizure occurrence. Correlation between number and type of anti-epileptic drugs (AED) and complement analytes was also performed. RESULTS: We found: CONCLUSION: This study adds to evidence implicating complement in pathogenesis of epilepsy and may allow the development of better therapeutics and prognostic markers in the future. Replication in a larger sample set is needed to validate the findings of the study.

Cerebrospinal fluid extracellular vesicle enrichment for protein biomarker discovery in neurological disease; multiple sclerosis.

The discovery of disease biomarkers, along with the use of "liquid biopsies" as a minimally invasive source of biomarkers, continues to be of great interest. In inflammatory diseases of the central nervous system (CNS), cerebrospinal fluid (CSF) is the most obvious biofluid source. Extracellular vesicles (EVs) are also present in CSF and are thought to be potential "biomarker treasure chests". However, isolating these CSF-derived EVs remains challenging. This small-scale pilot study developed and tested a protocol to enrich for CSF-EVs, both in relapsing remitting multiple sclerosis (RRMS) CSF and controls. These were subsequently compared, using an aptamer based proteomics array, SOMAscan™. EVs were enriched from RRMS patient (n = 4) and non-demyelinating control (idiopathic intracranial hypertension (IIH) (n = 3)) CSF using precipitation and mini size-exclusion chromatography (SEC). EV-enriched fractions were selected using pre-defined EV characteristics, including increased levels of tetraspanins. EVs and paired CSF were analysed by SOMAscan™, providing relative abundance data for 1128 proteins. CSF-EVs were characterised, revealing exosome-like features: rich in tetraspanins CD9 and CD81, size ~100 nm, and exosome-like morphology by TEM. Sufficient quantities of, SOMAscan™ compatible, EV material was obtained from 5 ml CSF for proteomics analysis. Overall, 348 and 580 proteins were identified in CSF-EVs and CSF, respectively, of which 50 were found to be significantly (t-test) and exclusively enriched in RRMS CSF-EVs. Selected proteins, Plasma kallikrein and Apolipoprotein-E4, were further validated by western blot and appeared increased in CSF-EVs compared to CSF. Functional enrichment analysis of the 50 enriched proteins revealed strong associations with biological processes relating to MS pathology and also extracellular regions, consistent with EV enrichment. This pilot study demonstrates practicality for EV enrichment in CSF derived from patients with MS and controls, allowing detailed analysis of protein profiles that may offer opportunities to identify novel biomarkers and therapeutic approaches in CNS inflammatory diseases.

Plasma complement biomarkers distinguish multiple sclerosis and neuromyelitis optica spectrum disorder.

BACKGROUND: Multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) are autoimmune inflammatory demyelinating diseases of the central nervous system. Although distinguished by clinicoradiological and demographic features, early manifestations can be similar complicating management. Antibodies against aquaporin-4 support the diagnosis of NMOSD but are negative in some patients. Therefore, there is unmet need for biomarkers that enable early diagnosis and disease-specific intervention. OBJECTIVE: We investigated whether plasma complement proteins are altered in MS and NMOSD and provide biomarkers that distinguish these diseases. METHODS: Plasma from 54 NMOSD, 40 MS and 69 control donors was tested in multiplex assays measuring complement activation products and proteins. Using logistic regression, we tested whether combinations of complement analytes distinguished NMOSD from controls and MS. RESULTS: All activation products were elevated in NMOSD compared to either control or MS. Four complement proteins (C1inh, C1s, C5 and FH) were higher in NMOSD compared to MS or controls. A model comprising C1inh and terminal complement complex (TCC) distinguished NMOSD from MS (area under the curve (AUC): 0.98), while C1inh and C5 distinguished NMOSD from controls (AUC: 0.94). CONCLUSION: NMOSD is distinguished from MS by plasma complement biomarkers. Selected complement analytes enable differential diagnosis. Findings support trials of anti-complement therapies in NMOSD.

Seroprevalence of Molluscum contagiosum virus in German and UK populations.

Molluscum contagiosum virus (MCV) is a significant but underreported skin pathogen for children and adults. Seroprevalence studies can help establish burden of disease. Enzyme linked immunosorbent assay (ELISA) based studies have been published for Australian and Japanese populations and the results indicate seroprevalences between 6 and 22 percent in healthy individuals, respectively. To investigate seroprevalence in Europe, we have developed a recombinant ELISA using a truncated MCV virion surface protein MC084 (V123-R230) expressed in E. coli. The ELISA was found to be sensitive and specific, with low inter- and intra-assay variability. Sera from 289 German adults and children aged 0-40 years (median age 21 years) were analysed for antibodies against MC084 by direct binding ELISA. The overall seropositivity rate was found to be 14.8%. The seropositivity rate was low in children below the age of one (4.5%), peaked in children aged 2-10 years (25%), and fell again in older populations (11-40 years; 12.5%). Ten out of 33 healthy UK individuals (30.3%; median age 27 years) had detectable MC084 antibodies. MCV seroconversion was more common in dermatological and autoimmune disorders, than in immunocompromised patients or in patients with multiple sclerosis. Overall MCV seroprevalence is 2.1 fold higher in females than in males in a UK serum collection. German seroprevalences determined in the MC084 ELISA (14.8%) are at least three times higher than incidence of MC in a comparable Swiss population (4.9%). While results are not strictly comparable, this is lower than Australian seroprevalence in a virion based ELISA (n = 357; 23%; 1999), but higher than the seroprevalence reported in a Japanese study using an N-terminal truncation of MC133 (n = 108, 6%; 2000. We report the first large scale serological survey of MC in Europe (n = 393) and the first MCV ELISA based on viral antigen expressed in E. coli.

Complement regulator factor H in multiple sclerosis.

A recent proteomic study published in this journal demonstrated lower cerebrospinal fluid (CSF) expression of factor H (fH), an important complement regulator, along with two other complement proteins, in active multiple sclerosis (MS) patients. We have previously demonstrated raised serum fH levels in MS and here, an extended analysis, quantifying fH in CSF, demonstrates no change in fH levels in active disease, but significantly raised levels in progressive disease. These findings support our previous work showing raised serum fH in patients with progressive MS, and our results predict that CSF fH levels will be raised rather than reduced in active disease.