The MOG antibody non-P42 epitope is predictive of a relapsing course in MOG antibody-associated disease.

BACKGROUND: Myelin oligodendrocyte glycoprotein (MOG) IgG seropositivity is a prerequisite for MOG antibody-associated disease (MOGAD) diagnosis. While a significant proportion of patients experience a relapsing disease, there is currently no biomarker predictive of disease course. We aim to determine whether MOG-IgG epitopes can predict a relapsing course in MOGAD patients. METHODS: MOG-IgG-seropositive confirmed adult MOGAD patients were included (n=202). Serum MOG-IgG and epitope binding were determined by validated flow cytometry live cell-based assays. Associations between epitopes, disease course, clinical phenotype, Expanded Disability Status Scale and Visual Functional System Score at onset and last review were evaluated. RESULTS: Of 202 MOGAD patients, 150 (74%) patients had MOG-IgG that recognised the immunodominant proline42 (P42) epitope and 115 (57%) recognised histidine103/serine104 (H103/S104). Fifty-two (26%) patients had non-P42 MOG-IgG and showed an increased risk of a relapsing course (HR 1.7; 95% CI 1.15 to 2.60, p=0.009). Relapse-freedom was shorter in patients with non-P42 MOG-IgG (p=0.0079). Non-P42 MOG-IgG epitope status remained unchanged from onset throughout the disease course and was a strong predictor of a relapsing course in patients with unilateral optic neuritis (HR 2.7, 95% CI 1.06 to 6.98, p=0.038), with high specificity (95%, 95% CI 77% to 100%) and positive predictive value (85%, 95% CI 45% to 98%). CONCLUSIONS: Non-P42 MOG-IgG predicts a relapsing course in a significant subgroup of MOGAD patients. Patients with unilateral optic neuritis, the most frequent MOGAD phenotype, can reliably be tested at onset, regardless of age and sex. Early detection and specialised management in these patients could minimise disability and improve long-term outcomes.

Infection and Vaccine Induced Spike Antibody Responses Against SARS-CoV-2 Variants of Concern in COVID-19-Naïve Children and Adults.

Although a more efficient adaptive humoral immune response has been proposed to underlie the usually favorable outcome of pediatric COVID-19, the breadth of viral and vaccine cross-reactivity toward the ever-mutating Spike protein among variants of concern (VOCs) has not yet been compared between children and adults. We assessed antibodies to conformational Spike in COVID-19-naïve children and adults vaccinated by BNT162b2 and ChAdOx1, and naturally infected with SARS-CoV-2 Early Clade, Delta, and Omicron. Sera were analyzed against Spike including naturally occurring VOCs Alpha, Beta, Gamma, Delta, and Omicron BA.1, BA.2, BA.5, BQ.1.1, BA2.75.2, and XBB.1, and variants of interest Epsilon, Kappa, Eta, D.2, and artificial mutant Spikes. There was no notable difference between breadth and longevity of antibody against VOCs in children and adults. Vaccinated individuals displayed similar immunoreactivity profiles across variants compared with naturally infected individuals. Delta-infected patients had an enhanced cross-reactivity toward Delta and earlier VOCs compared to patients infected by Early Clade SARS-CoV-2. Although Omicron BA.1, BA.2, BA.5, BQ.1.1, BA2.75.2, and XBB.1 antibody titers were generated after Omicron infection, cross-reactive binding against Omicron subvariants was reduced across all infection, immunization, and age groups. Some mutations, such as 498R and 501Y, epistatically combined to enhance cross-reactive binding, but could not fully compensate for antibody-evasive mutations within the Omicron subvariants tested. Our results reveal important molecular features central to the generation of high antibody titers and broad immunoreactivity that should be considered in future vaccine design and global serosurveillance in the context of limited vaccine boosters available to the pediatric population.

Tracking the clonal dynamics of SARS-CoV-2-specific T cells in children and adults with mild/asymptomatic COVID-19.

Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV-2-specific naïve T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. A novel population of naïve interferon-activated T cells is expanded in acute COVID-19 and is recruited into the memory compartment during convalescence in adults but not children. This was associated with the development of robust CD4+ memory T cell responses in adults but not children. These data suggest that rapid clearance of SARS-CoV-2 in children may compromise their cellular immunity and ability to resist reinfection.

Validation of a Flow Cytometry Live Cell-Based Assay to Detect Myelin Oligodendrocyte Glycoprotein Antibodies for Clinical Diagnostics.

BACKGROUND: Myelin oligodendrocyte glycoprotein antibodies (MOG Ab) are essential in the diagnosis of MOG Ab-associated disease (MOGAD). Live cell-based assays (CBAs) are the gold standard for MOG Ab detection with improved sensitivity and specificity over fixed CBAs. A number of testing centers have used flow cytometry for its high throughput and quantitative utility. Presently, there is increasing demand to translate these research-based methods into an accredited routine diagnostic setting. METHODS: A flow cytometry live CBA was used to detect MOG Ab in patients with demyelination. Serostatuses were compared between a research-based assay and a streamlined diagnostic assay. Inter-laboratory validation of the streamlined assay was performed in an accredited diagnostic laboratory. Further streamlining was performed by introducing a borderline serostatus range and reducing the number of controls used to determine the positivity threshold. RESULTS: High serostatus agreement (98%-100%) was observed between streamlined and research-based assays. Intra- and inter-assay imprecision was improved in the streamlined assay (mean intra- and inter-assay CV = 7.3% and 27.8%, respectively) compared to the research-based assay (mean intra- and inter-assay CV = 11.8% and 33.6%, respectively). Borderline positive and clear positive serostatuses were associated with confirmed phenotypes typical of MOGAD. Compared to using 24 controls, robust serostatus classification was observed when using 13 controls without compromising analytical performance (93%-98.5% agreement). CONCLUSIONS: Flow cytometry live CBAs show robust utility in determining MOG Ab serostatus. Streamlining and standardizing use of this assay for diagnostics would improve the accuracy and reliability of routine testing to aid diagnosis and treatment of patients with demyelination.

SARS-CoV-2 neutralizing antibodies: Longevity, breadth, and evasion by emerging viral variants.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus-cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)-confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of "high responders" maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design.

Effects of the Positive Threshold and Data Analysis on Human MOG Antibody Detection by Live Flow Cytometry.

Human autoantibodies targeting myelin oligodendrocyte glycoprotein (MOG Ab) have become a useful clinical biomarker for the diagnosis of a spectrum of inflammatory demyelinating disorders. Live cell-based assays that detect MOG Ab against conformational MOG are currently the gold standard. Flow cytometry, in which serum binding to MOG-expressing cells and control cells are quantitively evaluated, is a widely used observer-independent, precise, and reliable detection method. However, there is currently no consensus on data analysis; for example, seropositive thresholds have been reported using varying standard deviations above a control cohort. Herein, we used a large cohort of 482 sera including samples from patients with monophasic or relapsing demyelination phenotypes consistent with MOG antibody-associated demyelination and other neurological diseases, as well as healthy controls, and applied a series of published analyses involving a background subtraction (delta) or a division (ratio). Loss of seropositivity and reduced detection sensitivity were observed when MOG ratio analyses or when 10 standard deviation (SD) or an arbitrary number was used to establish the threshold. Background binding and MOG ratio value were negatively correlated, in which patients seronegative by MOG ratio had high non-specific binding, a characteristic of serum that must be acknowledged. Most MOG Ab serostatuses were similar across analyses when optimal thresholds obtained by ROC analyses were used, demonstrating the robust nature and high discriminatory power of flow cytometry cell-based assays. With increased demand to identify MOG Ab-positive patients, a consensus on analysis is vital to improve patient diagnosis and for cross-study comparisons to ultimately define MOG Ab-associated disorders.

Characterization of the human myelin oligodendrocyte glycoprotein antibody response in demyelination.

Over recent years, human autoantibodies targeting myelin oligodendrocyte glycoprotein (MOG Ab) have been associated with monophasic and relapsing central nervous system demyelination involving the optic nerves, spinal cord, and brain. While the clinical relevance of MOG Ab detection is becoming increasingly clear as therapeutic and prognostic differences from multiple sclerosis are acknowledged, an in-depth characterization of human MOG Ab is required to answer key challenges in patient diagnosis, treatment, and prognosis. Herein, we investigated the epitope, binding sensitivity, and affinity of MOG Ab in a cohort of 139 and 148 MOG antibody-seropositive children and adults (n = 287 patients at baseline, 130 longitudinal samples, and 22 cerebrospinal fluid samples). MOG extracellular domain was also immobilized to determine the affinity of MOG Ab. MOG Ab response was of immunoglobulin G1 isotype, and was of peripheral rather than intrathecal origin. High affinity MOG Ab were detected in 15% paediatric and 18% adult sera. More than 75% of paediatric and adult MOG Ab targeted a dominant extracellular antigenic region around Proline42. MOG Ab titers fluctuated over the progression of disease, but affinity and reactivity to Proline42 remained stable. Adults with a relapsing course intrinsically presented with a reduced immunoreactivity to Proline42 and had a more diverse MOG Ab response, a feature that may be harnessed for predicting relapse. Higher titers of MOG Ab were observed in more severe phenotypes and during active disease, supporting the pathogenic role of MOG Ab. Loss of MOG Ab seropositivity was observed upon conformational changes to MOG, and this greatly impacted the sensitivity of the detection of relapsing disorders, largely considered as more severe. Careful consideration of the binding characteristics of autoantigens should be taken into account when detecting disease-relevant autoantibodies.

The Effect of ACTN3 Gene Doping on Skeletal Muscle Performance.

Loss of expression of ACTN3, due to homozygosity of the common null polymorphism (p.Arg577X), is underrepresented in elite sprint/power athletes and has been associated with reduced muscle mass and strength in humans and mice. To investigate ACTN3 gene dosage in performance and whether expression could enhance muscle force, we performed meta-analysis and expression studies. Our general meta-analysis using a Bayesian random effects model in elite sprint/power athlete cohorts demonstrated a consistent homozygous-group effect across studies (per allele OR = 1.4, 95% CI 1.3-1.6) but substantial heterogeneity in heterozygotes. In mouse muscle, rAAV-mediated gene transfer overexpressed and rescued α-actinin-3 expression. Contrary to expectation, in vivo "doping" of ACTN3 at low to moderate doses demonstrated an absence of any change in function. At high doses, ACTN3 is toxic and detrimental to force generation, to demonstrate gene doping with supposedly performance-enhancing isoforms of sarcomeric proteins can be detrimental for muscle function. Restoration of α-actinin-3 did not enhance muscle mass but highlighted the primary role of α-actinin-3 in modulating muscle metabolism with altered fatiguability. This is the first study to express a Z-disk protein in healthy skeletal muscle and measure the in vivo effect. The sensitive balance of the sarcomeric proteins and muscle function has relevant implications in areas of gene doping in performance and therapy for neuromuscular disease.

How does α-actinin-3 deficiency alter muscle function? Mechanistic insights into ACTN3, the 'gene for speed'.

An estimated 1.5 billion people worldwide are deficient in the skeletal muscle protein α-actinin-3 due to homozygosity for the common ACTN3 R577X polymorphism. α-Actinin-3 deficiency influences muscle performance in elite athletes and the general population. The sarcomeric α-actinins were originally characterised as scaffold proteins at the muscle Z-line. Through studying the Actn3 knockout mouse and α-actinin-3 deficient humans, significant progress has been made in understanding how ACTN3 genotype alters muscle function, leading to an appreciation of the diverse roles that α-actinins play in muscle. The α-actinins interact with a number of partner proteins, which broadly fall into three biological pathways-structural, metabolic and signalling. Differences in functioning of these pathways have been identified in α-actinin-3 deficient muscle that together contributes to altered muscle performance in mice and humans. Here we discuss new insights that have been made in understanding the molecular mechanisms that underlie the consequences of α-actinin-3 deficiency.