IL-1 receptor 1 signaling shapes the development of viral antigen-specific CD4+ T cell responses following COVID-19 mRNA vaccination.

BACKGROUND: The innate immune cytokine interleukin (IL)-1 can affect T cell immunity, a critical factor in host defense. In a previous study, we identified a subset of human CD4+ T cells which express IL-1 receptor 1 (IL-1R1). However, the expression of such receptor by viral antigen-specific CD4+ T cells and its biological implication remain largely unexplored. This led us to investigate the implication of IL-1R1 in the development of viral antigen-specific CD4+ T cell responses in humans, including healthy individuals and patients with primary antibody deficiency (PAD), and animals. METHODS: We characterized CD4+ T cells specific for SARS-CoV-2 spike (S) protein, influenza virus, and cytomegalovirus utilizing multiplexed single cell RNA-seq, mass cytometry and flow cytometry followed by an animal study. FINDINGS: In healthy individuals, CD4+ T cells specific for viral antigens, including S protein, highly expressed IL-1R1. IL-1ß promoted interferon (IFN)-γ expression by S protein-stimulated CD4+ T cells, supporting the functional implication of IL-1R1. Following the 2nd dose of COVID-19 mRNA vaccines, S protein-specific CD4+ T cells with high levels of IL-1R1 increased, likely reflecting repetitive antigenic stimulation. The expression levels of IL-1R1 by such cells correlated with the development of serum anti-S protein IgG antibody. A similar finding of increased expression of IL-1R1 by S protein-specific CD4+ T cells was also observed in patients with PAD following COVID-19 mRNA vaccination although the expression levels of IL-1R1 by such cells did not correlate with the levels of serum anti-S protein IgG antibody. In mice immunized with COVID-19 mRNA vaccine, neutralizing IL-1R1 decreased IFN-γ expression by S protein-specific CD4+ T cells and the development of anti-S protein IgG antibody. INTERPRETATION: Our results demonstrate the significance of IL-1R1 expression in CD4+ T cells for the development of viral antigen-specific CD4+ T cell responses, contributing to humoral immunity. This provides an insight into the regulation of adaptive immune responses to viruses via the IL-1 and IL-1R1 interface. FUNDING: Moderna to HJP, National Institutes of Health (NIH) 1R01AG056728 and R01AG055362 to IK and KL2 TR001862 to JJS, Quest Diagnostics to IK and RB, and the Mathers Foundation to RB.

Clinical utility of plasma Aß42/40 ratio by LC-MS/MS in Alzheimer's disease assessment.

Introduction: Plasma Aß42/40 ratio can help predict amyloid PET status, but its clinical utility in Alzheimer's disease (AD) assessment is unclear. Methods: Aß42/40 ratio was measured by LC-MS/MS for 250 specimens with associated amyloid PET imaging, diagnosis, and demographic data, and for 6,192 consecutive clinical specimens submitted for Aß42/40 testing. Results: High diagnostic sensitivity and negative predictive value (NPV) for Aß-PET positivity were observed, consistent with the clinical performance of other plasma LC-MS/MS assays, but with greater separation between Aß42/40 values for individuals with positive vs. negative Aß-PET results. Assuming a moderate prevalence of Aß-PET positivity, a cutpoint was identified with 99% NPV, which could help predict that AD is likely not the cause of patients' cognitive impairment and help reduce PET evaluation by about 40%. Conclusion: High-throughput plasma Aß42/40 LC-MS/MS assays can help identify patients with low likelihood of AD pathology, which can reduce PET evaluations, allowing for cost savings.

Machine Learning Analysis Using RNA Sequencing to Distinguish Neuromyelitis Optica from Multiple Sclerosis and Identify Therapeutic Candidates.

This study aims to identify RNA biomarkers distinguishing neuromyelitis optica (NMO) from relapsing-remitting multiple sclerosis (RRMS) and explore potential therapeutic applications leveraging machine learning (ML). An ensemble approach was developed using differential gene expression analysis and competitive ML methods, interrogating total RNA-sequencing data sets from peripheral whole blood of treatment-naïve patients with RRMS and NMO and healthy individuals. Pathway analysis of candidate biomarkers informed the biological context of disease, transcription factor activity, and small-molecule therapeutic potential. ML models differentiated between patients with NMO and RRMS, with the performance of certain models exceeding 90% accuracy. RNA biomarkers driving model performance were associated with ribosomal dysfunction and viral infection. Regulatory networks of kinases and transcription factors identified biological associations and identified potential therapeutic targets. Small-molecule candidates capable of reversing perturbed gene expression were uncovered. Mitoxantrone and vorinostat-two identified small molecules with previously reported use in patients with NMO and experimental autoimmune encephalomyelitis-reinforced discovered expression signatures and highlighted the potential to identify new therapeutic candidates. Putative RNA biomarkers were identified that accurately distinguish NMO from RRMS and healthy individuals. The application of multivariate approaches in analysis of RNA-sequencing data further enhances the discovery of unique RNA biomarkers, accelerating the development of new methods for disease detection, monitoring, and therapeutics. Integrating biological understanding further enhances detection of disease-specific signatures and possible therapeutic targets.

New plasma LC-MS/MS assays for the quantitation of beta-amyloid peptides and identification of apolipoprotein E proteoforms for Alzheimer's disease risk assessment.

Early detection of Alzheimer's disease (AD) represents an unmet clinical need. Beta-amyloid (Aß) plays an important role in AD pathology, and the Aß42/40 peptide ratio is a good indicator for amyloid deposition. In addition, variants of the apolipoprotein E (APOE) gene are associated with variable AD risk. Here, we describe the development and validation of high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for plasma Aß40 and Aß42 quantitation, as well as apolipoprotein E (ApoE) proteotype determination as a surrogate for APOE genotype. Aß40 and Aß42 were simultaneously immunoprecipitated from plasma, proteolytically digested, and quantitated by LC-MS/MS. ApoE proteotype status was qualitatively assessed by targeting tryptic peptides from the ApoE2, ApoE3, and ApoE4 proteoforms. Both assays were validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Within-run precision was 1.8%-4.2% (Aß40), 1.9%-7.2% (Aß42), and 2.6%-8.3% (Aß42/40 ratio). Between-run precision was 3.5%-5.9% (Aß40), 3.8%-8.0% (Aß42), and 3.3%-8.7% (Aß42/40 ratio). Both Aß40 and Aß42 were linear from 10 to 2500 pg/mL. Identified ApoE proteotypes had 100% concordance with APOE genotypes. We have developed a precise, accurate, and sensitive high-throughput LC-MS/MS assay for plasma Aß40, Aß42, and proteoforms of ApoE.

Prior cycles of anti-CD20 antibodies affect antibody responses after repeated SARS-CoV-2 mRNA vaccination.

BACKGROUNDWhile B cell depletion is associated with attenuated antibody responses to SARS-CoV-2 mRNA vaccination, responses vary among individuals. Thus, elucidating the factors that affect immune responses after repeated vaccination is an important clinical need.METHODSWe evaluated the quality and magnitude of the T cell, B cell, antibody, and cytokine responses to a third dose of BNT162b2 or mRNA-1273 mRNA vaccine in patients with B cell depletion.RESULTSIn contrast with control individuals (n = 10), most patients on anti-CD20 therapy (n = 48) did not demonstrate an increase in spike-specific B cells or antibodies after a third dose of vaccine. A third vaccine elicited significantly increased frequencies of spike-specific non-naive T cells. A small subset of B cell-depleted individuals effectively produced spike-specific antibodies, and logistic regression models identified time since last anti-CD20 treatment and lower cumulative exposure to anti-CD20 mAbs as predictors of those having a serologic response. B cell-depleted patients who mounted an antibody response to 3 vaccine doses had persistent humoral immunity 6 months later.CONCLUSIONThese results demonstrate that serial vaccination strategies can be effective for a subset of B cell-depleted patients.FUNDINGThe NIH (R25 NS079193, P01 AI073748, U24 AI11867, R01 AI22220, UM 1HG009390, P01 AI039671, P50 CA121974, R01 CA227473, U01CA260507, 75N93019C00065, K24 AG042489), NIH HIPC Consortium (U19 AI089992), the National Multiple Sclerosis Society (CA 1061-A-18, RG-1802-30153), the Nancy Taylor Foundation for Chronic Diseases, Erase MS, and the Claude D. Pepper Older Americans Independence Center at Yale (P30 AG21342).

Microfluidic Immuno-Serolomic Assay Reveals Systems Level Association with COVID-19 Pathology and Vaccine Protection.

How to develop highly informative serology assays to evaluate the quality of immune protection against coronavirus disease-19 (COVID-19) has been a global pursuit over the past years. Here, a microfluidic high-plex immuno-serolomic assay is developed to simultaneously measure50 plasma or serum samples for50 soluble markers including 35proteins, 11 anti-spike/receptor binding domian (RBD) IgG antibodies spanningmajor variants, and controls. This assay demonstrates the quintuplicate test in a single run with high throughput, low sample volume, high reproducibilityand accuracy. It is applied to the measurement of 1012 blood samples including in-depth analysis of sera from 127 patients and 21 healthy donors over multiple time points, either with acute COVID infection or vaccination. The protein analysis reveals distinct immune mediator modules that exhibit a reduced degree of diversity in protein-protein cooperation in patients with hematologic malignancies or receiving B cell depletion therapy. Serological analysis identifies that COVID-infected patients with hematologic malignancies display impaired anti-RBD antibody response despite high level of anti-spike IgG, which can be associated with limited clonotype diversity and functional deficiency in B cells. These findings underscore the importance to individualize immunization strategies for these high-risk patients and provide an informative tool to monitor their responses at the systems level.

Multiple Sclerosis Followed by Neuromyelitis Optica Spectrum Disorder: From the National Multiple Sclerosis Society Case Conference Proceedings.

A woman presented at age 18 years with partial myelitis and diplopia and experienced multiple subsequent relapses. Her MRI demonstrated T2 abnormalities characteristic of multiple sclerosis (MS) (white matter ovoid lesions and Dawson fingers), and CSF demonstrated an elevated IgG index and oligoclonal bands restricted to the CSF. Diagnosed with clinically definite relapsing-remitting MS, she was treated with various MS disease-modifying therapies and eventually began experiencing secondary progression. At age 57 years, she developed an acute longitudinally extensive transverse myelitis and was found to have AQP4 antibodies by cell-based assay. Our analysis of the clinical course, radiographic findings, molecular diagnostic methods, and treatment response characteristics support the hypothesis that our patient most likely had 2 CNS inflammatory disorders: MS, which manifested as a teenager, and neuromyelitis optica spectrum disorder, which evolved in her sixth decade of life. This case emphasizes a key principle in neurology practice, which is to reconsider whether the original working diagnosis remains tenable, especially when confronted with evidence (clinical and/or paraclinical) that raises the possibility of a distinctively different disorder.

Clinical utility of plasma Aß42/40 ratio by LC-MS/MS in Alzheimer's disease assessment.

INTRODUCTION: Plasma Aß42/40 ratio can be used to help predict amyloid PET status, but its clinical utility in Alzheimer's disease (AD) assessment is unclear. METHODS: Aß42/40 ratio was measured by LC-MS/MS in 250 specimens with associated amyloid PET imaging, diagnosis, and demographic data, and 6,192 consecutive clinical specimens submitted for Aß42/40 testing. RESULTS: High diagnostic sensitivity and negative predictive value (NPV) for Aß-PET positivity were observed, consistent with the clinical performance of other plasma LC-MS/MS assays, but with greater separation between Aß42/40 values for individuals with positive vs negative Aß-PET results. Assuming a moderate prevalence of Aß-PET positivity, a cutpoint was identified with 99% NPV, which could help predict that AD is likely not the cause of patients' cognitive impairment and help reduce PET evaluation by about 40%. DISCUSSION: Using high-throughput plasma Aß42/40 LC-MS/MS assays can help reduce PET evaluations in patients with low likelihood of AD pathology, allowing for cost savings.

Microfluidic immuno-serology assay revealed a limited diversity of protection against COVID-19 in patients with altered immunity.

The immune response to SARS-CoV-2 for patients with altered immunity such as hematologic malignancies and autoimmune disease may differ substantially from that in general population. These patients remain at high risk despite wide-spread adoption of vaccination. It is critical to examine the differences at the systems level between the general population and the patients with altered immunity in terms of immunologic and serological responses to COVID-19 infection and vaccination. Here, we developed a novel microfluidic chip for high-plex immuno-serological assay to simultaneously measure up to 50 plasma or serum samples for up to 50 soluble markers including 35 plasma proteins, 11 anti-spike/RBD IgG antibodies spanning all major variants, and controls. Our assay demonstrated the quintuplicate test in a single run with high throughput, low sample volume input, high reproducibility and high accuracy. It was applied to the measurement of 1,012 blood samples including in-depth analysis of sera from 127 patients and 21 healthy donors over multiple time points, either with acute COVID infection or vaccination. The protein association matrix analysis revealed distinct immune mediator protein modules that exhibited a reduced degree of diversity in protein-protein cooperation in patients with hematologic malignancies and patients with autoimmune disorders receiving B cell depletion therapy. Serological analysis identified that COVID infected patients with hematologic malignancies display impaired anti-RBD antibody response despite high level of anti-spike IgG, which could be associated with limited clonotype diversity and functional deficiency in B cells and was further confirmed by single-cell BCR and transcriptome sequencing. These findings underscore the importance to individualize immunization strategy for these high-risk patients and provide an informative tool to monitor their responses at the systems level.

Changes in ganglioside antibody positivity rates during the COVID-19 pandemic.

Reports suggested an association between SARS-CoV-2 infection and GBS, but subsequent studies produced conflicting results regarding the incidence of GBS during the pandemic. This study assessed positivity rates for GQ1b, GM-1, GD1a, and GD1b for tests performed January 2016, through March 2021, at a national laboratory. Relative to pre-pandemic levels, positivity rates during the pandemic declined by 61% for GQ1b and 24% for GM-1, while unchanged for GD1a and GD1b. These findings suggest heterogeneity with positivity rates of GBS-associated ganglioside antibodies during the COVID-19 pandemic. Mitigation strategies during the pandemic may have reduced the frequency of certain forms of GBS.

Progressive multifocal leukoencephalopathy in dimethyl fumarate-treated multiple sclerosis patients.

Dimethyl fumarate (DMF), a fumaric acid with antioxidant and immunomodulatory properties, is among the most commonly used oral therapies for relapsing multiple sclerosis (MS). Progressive multifocal leukoencephalopathy (PML) has been associated with several disease-modifying therapies (DMTs), including DMF in treating MS. We present detailed clinical characteristics of nine PML cases and show that the PML incidence in DMF-treated patients is 0.02 per 1000 patients. In addition to persistent severe lymphopenia, older age appears to be a potential risk for PML. However, younger patients without lymphopenia were also observed to develop PML. DMF-associated PML has occurred in patients with absolute lymphocyte counts (ALCs) above the guideline threshold, suggesting that changes in specific subsets might be more important than total ALC. Furthermore, since DMF has been found to decrease immune cell migration by decreasing the expression of adhesive molecules, the cerebrospinal fluid (CSF) immune profile may also be useful for assessing PML risk in DMF-treated patients. This review provides an up-to-date assessment of PML cases occurring in DMF-treated patients and discusses other potential considerations in light of our current understanding of DMF's mechanism of action on the immune system in the periphery and in the central nervous system (CNS).

COVID-19 Vaccination Reactogenicity in Persons With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: There are limited data on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine reactogenicity in persons with multiple sclerosis (PwMS) and how reactogenicity is affected by disease-modifying therapies (DMTs). The objective of this retrospective cross-sectional study was to generate real-world multiple sclerosis-specific vaccine safety information, particularly in the context of specific DMTs, and provide information to mitigate specific concerns in vaccine hesitant PwMS. METHODS: Between 3/2021 and 6/2021, participants in iConquerMS, an online people-powered research network, reported SARS-CoV-2 vaccines, experiences of local (itch, pain, redness, swelling, or warmth at injection site) and systemic (fever, chills, fatigue, headache, joint pain, malaise, muscle ache, nausea, allergic, and other) reactions within 24 hours (none, mild, moderate, and severe), DMT use, and other attributes. Multivariable models characterized associations between clinical factors and reactogenicity. RESULTS: In 719 PwMS, 64% reported experiencing a reaction after their first vaccination shot, and 17% reported a severe reaction. The most common reactions were pain at injection site (54%), fatigue (34%), headache (28%), and malaise (21%). Younger age, being female, prior SARS-CoV-2 infection, and receiving the ChAdOx1 nCoV-19 (Oxford-AstraZeneca) vs BNT162b2 (Pfizer-BioNTech) vaccine were associated with experiencing a reaction after the first vaccine dose. Similar relationships were observed for a severe reaction, including higher odds of reactions among PwMS with more physical impairment and lower odds of reactions for PwMS on an alpha4-integrin blocker or sphingosine-1-phosphate receptor modulator. In 442 PwMS who received their second vaccination shot, 74% reported experiencing a reaction, whereas 22% reported a severe reaction. Reaction profiles after the second shot were similar to those reported after the first shot. Younger PwMS and those who received the mRNA-1273 (Moderna) vs BNT162b2 vaccine reported higher reactogenicity after the second shot, whereas those on a sphingosine-1-phosphate receptor modulator or fumarate were significantly less likely to report a reaction. DISCUSSION: SARS-CoV-2 vaccine reactogenicity profiles and the associated factors in this convenience sample of PwMS appear similar to those reported in the general population. PwMS on specific DMTs were less likely to report vaccine reactions. Overall, the short-term vaccine reactions experienced in the study population were mostly self-limiting, including pain at the injection site, fatigue, headache, and fever.

B cell depletion changes the immune cell profile in multiple sclerosis patients: One-year report.

B cell depletion therapy has been shown to be beneficial in multiple sclerosis (MS). However, the mechanism by which B cell depletion mediates its beneficial effects in MS is still unclear. To better understand how B cell depletion may benefit patients with a disease previously thought to be primarily mediated by CD4 T cells, immune profiles were monitored in 48 patients in a phase II trial of ublituximab, a glycoengineered CD20 monoclonal antibody, at 18 time points over a year. As we previously described there was a significant shift in the percentages of T cells, NK cells, and myeloid cells following the initial dose of ublituximab, but this shift normalized within a week and these populations remained stable for the duration of the study. However, T cell subsets changed with an increase in the percentage of naïve CD4 and CD8 T cells and a decline in memory T cells. Importantly, the percentage of Th1 and CD4+GM-CSF+ T cells decreased, while the percentage of Tregs continued to increase over the year. Ublituximab not only depleted CD20+ B cells, but also CD20+ T cells. The favorable changes in the T cell subsets may contribute to the beneficial effects of B cell depletion therapy.

Brain volume change after high-dose immunosuppression and autologous hematopoietic cell transplantation for relapsing-remitting multiple sclerosis.

BACKGROUND: Brain volume loss (BVL) is commonly observed after high-dose immunosuppression and autologous hematopoietic cell transplantation (HDIT/HCT) for treatment of multiple sclerosis (MS). To better understand the mechanisms of underlying BVL associated with this treatment, we characterized the time courses of whole-brain (WB), grey-matter (GM) and white-matter (WM) volume loss in relapsing-remitting MS (RRMS) patients who received BEAM-based HDIT/HCT. METHODS: We used Jacobian integration to measure MRI-based WB, GM and WM volume changes up to 5 years after transplant in twenty-four RRMS participants who underwent BEAM-based HDIT/HCT. Using a two-piecewise mixed-effects model, we estimated the short-term (baseline to 1 year) and long-term (beyond 1 year) rates of BVL after HDIT/HCT. We also compared the rates based on the presence of gadolinium-enhancing lesions at baseline, and the maintenance of event-free survival during follow-up. RESULTS: On average, accelerated short-term BVL of -1.37% (SE: 0.21), -0.86% (SE: 0.28) and -2.18% (SE: 0.26) occurred in WB, GM and WM, respectively. Baseline T1-weighted MRI WM lesion volume was a significant predictor in the WB (short-term) and the WM (short-term and long-term). The average rates of BVL after the initial acceleration were -0.22%/y (SE: 0.10), -0.13%/y (SE: 0.11) and -0.36%/y (SE: 0.11) in the WB, GM and WM, respectively. Participants with gadolinium-enhancing lesions at baseline had significantly higher short-term rates of GM (-1.56% vs. -0.27%, p = 0.01) and WB volume loss (-1.94% vs. -0.81%, p = 0.006) at 1 year follow-up as compared to those without gadolinium-enhancing lesions. WM volume loss was not significantly different (-2.59% vs. -1.66%, p = 0.16). Participants who maintained event-free survival had similar rates of BVL compared to those who did not. CONCLUSIONS: BVL may accelerate for months after HDIT/HCT. However, over the long-term, adequate HDIT/HCT may reduce BVL rates to those similar to normal aging at the WB level.

A STAT3 inhibitor ameliorates CNS autoimmunity by restoring Teff:Treg balance.

Reestablishing an appropriate balance between T effector cells (Teff) and Tregs is essential for correcting autoimmunity. Multiple sclerosis (MS) is an immune-mediated chronic CNS disease characterized by neuroinflammation, demyelination, and neuronal degeneration, in which the Teff:Treg balance is skewed toward pathogenic Teffs Th1 and Th17 cells. STAT3 is a key regulator of Teff:Treg balance. Using the structure-based design, we have developed a potentially novel small-molecule prodrug LLL12b that specifically inhibits STAT3 and suppresses Th17 differentiation and expansion. Moreover, LLL12b regulates the fate decision between Th17 and Tregs in an inflammatory environment, shifting Th17:Treg balance toward Tregs and favoring the resolution of inflammation. Therapeutic administration of LLL12b after disease onset significantly suppresses disease progression in adoptively transferred, chronic, and relapsing-remitting experimental autoimmune encephalomyelitis. Disease relapses were also significantly suppressed by LLL12b given during the remission phase. Additionally, LLL12b shifts Th17:Treg balance of CD4+ T cells from MS patients toward Tregs and increases Teff sensitivity to Treg-mediated suppression. These data suggest that selective inhibition of STAT3 by the small molecule LLL12b recalibrates the effector and regulatory arms of CD4+ T responses, representing a potentially clinically translatable therapeutic strategy for MS.

Pain in Multiple Sclerosis: Understanding Pathophysiology, Diagnosis, and Management Through Clinical Vignettes.

Neuropathic pain and other pain syndromes occur in the vast majority of patients with multiple sclerosis at some time during their disease course. Pain can become chronic and paroxysmal. In this review, we will utilize clinical vignettes to describe various pain syndromes associated with multiple sclerosis and their pathophysiology. These syndromes vary from central neuropathic pain or Lhermitte's phenomenon associated with central nervous system lesions to trigeminal neuralgia and optic neuritis pain associated with nerve lesions. Muscular pain can also arise due to spasticity. In addition, we will discuss strategies utilized to help patients manage these symptoms.

B cell depletion with ublituximab reshapes the T cell profile in multiple sclerosis patients.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system, thought to be mediated by myelin-specific CD4+ T cells. However, B cell depletion has proven to be an effective therapy for MS, but the mechanism is not well understood. This study was designed to determine how B cell depletion changes lymphocyte profiles. During a phase IIa clinical trial with ublituximab, a novel CD20 antibody, blood was collected from 48 MS patients at 11 time points over 24 weeks and the lymphocyte profiles were analyzed by flow cytometry. The percentage of naïve CD4+ and CD8+ T cells increased, while the percentage of both effector and central memory T cells declined. CD4+ Th1 effector cells decreased, while there was a significant increase in CD4+ regulatory T cells. The depletion of B cells had a favorable shift in the lymphocyte landscape, reducing the number of naïve T cells becoming activated and transitioning to memory T cells. The ratio of Th1 cells to CD4+ regulatory T cells declined, suggesting that immune regulation was being restored. These data suggest that loss of B cells as antigen presenting cells is a major mechanism of action for the beneficial effects of CD20 antibody therapy in MS.

Ocrelizumab infusion experience in patients with relapsing and primary progressive multiple sclerosis: Results from the phase 3 randomized OPERA I, OPERA II, and ORATORIO studies.

BACKGROUND: Ocrelizumab is an infusible humanized monoclonal antibody that selectively depletes CD20+ B cells. Infusion-related reactions (IRRs) were summarized from the OPERA I, OPERA II, and ORATORIO trials for relapsing and primary progressive multiple sclerosis (MS). METHODS: OPERA I and OPERA II were identical, randomized, double-blind, active-controlled trials that enrolled patients with relapsing MS (RMS). Patients in the ocrelizumab group initially received two 300-mg intravenous (IV) infusions separated by 14 days (on Days 1 and 15); subsequent doses were administered as single 600-mg IV infusions. Ocrelizumab-treated patients also received subcutaneous (SC) placebo injections 3 times weekly. Patients in the active comparator group received SC injections of IFN ß-1a 3 times weekly, as well as placebo infusions on Days 1 and 15 and Weeks 24, 48, and 72. ORATORIO was a randomized, parallel-group, double-blind, placebo-controlled study that enrolled patients with primary progressive MS (PPMS). As in the OPERA studies, patients in the ocrelizumab group initially received two 300-mg infusions separated by 14 days; however, ORATORIO patients continued to receive this divided-dose regimen throughout the study. The ORATORIO control group received IV placebo. Prior to each infusion, all patients in the OPERA and ORATORIO studies were pretreated with 100 mg IV methylprednisolone; additional prophylactic treatment with analgesics, antipyretics, and/or an IV or oral antihistamine was optional. IRRs were defined as adverse events that occurred during or within 24 h of IV infusion of ocrelizumab or placebo. RESULTS: Safety analyses included 1651 patients with RMS from OPERA I and OPERA II (ocrelizumab, n = 825; IFN ß-1a, n = 826) and 725 patients with PPMS from ORATORIO (ocrelizumab, n = 486; placebo, n = 239). Across studies, IRRs were reported in 34.3% (vs 9.7% with IFN ß-1a) and 39.9% (vs 25.5% with placebo) of ocrelizumab-treated patients in the pooled OPERA and ORATORIO populations, respectively. The majority of IRRs were mild to moderate in the OPERA (ocrelizumab, 92.6%; IFN ß-1a, 98.8%) and ORATORIO (ocrelizumab, 96.9%; placebo, 93.4%) studies. IRRs most commonly occurred with the first infusion. Severe IRRs were reported in 2.4% of ocrelizumab-treated patients in the OPERA studies (vs 0.1% with IFN ß-1a) and 1.2% of ocrelizumab-treated patients in ORATORIO (vs 1.7% with placebo). Two serious IRRs occurred across the OPERA studies, both of which occurred with the initial infusion. The first event occurred in an IFN ß-1a-treated patient in association with the initial infusion of IV placebo and consisted of severe balance disorder, dizziness, flushing, and hypoesthesia. The second event was a life-threatening reaction (bronchospasm) that occurred in an ocrelizumab-treated patient 15 min after the infusion started. Frequently reported IRR symptoms included pruritus, rash, throat irritation, and flushing. Premedication use, particularly antihistamines, was associated with fewer IRRs. CONCLUSION: Findings from the OPERA I, OPERA II, and ORATORIO trials show that IRRs were the most frequently reported adverse events with ocrelizumab, were mostly mild to moderate in severity, were reduced with appropriate pretreatment, and decreased with subsequent dosing. IRRs that did occur were effectively managed through infusion rate adjustment and symptomatic treatment.