Converging synaptic and network dysfunctions in distinct autoimmune encephalitis.

Psychiatric and neurological symptoms, as well as cognitive deficits, represent a prominent phenotype associated with variable forms of autoimmune encephalitis, regardless of the neurotransmitter receptor targeted by autoantibodies. The mechanistic underpinnings of these shared major neuropsychiatric symptoms remain however unclear. Here, we investigate the impacts of patient-derived monoclonal autoantibodies against the glutamatergic NMDAR (NMDAR mAb) and inhibitory GABAaR (GABAaR mAb) signalling in the hippocampal network. Unexpectedly, both excitatory and inhibitory synaptic receptor membrane dynamics, content and transmissions are altered by NMDAR or GABAaR mAb, irrespective of the affinity or antagonistic effect of the autoantibodies. The effect of NMDAR mAb on inhibitory synapses and GABAaR mAb on excitatory synapses requires neuronal activity and involves protein kinase signalling. At the cell level, both autoantibodies increase the excitation/inhibition balance of principal cell inputs. Furthermore, NMDAR or GABAaR mAb leads to hyperactivation of hippocampal networks through distinct alterations of principal cell and interneuron properties. Thus, autoantibodies targeting excitatory NMDAR or inhibitory GABAaR trigger convergent network dysfunctions through a combination of shared and distinct mechanisms.

Cross-regulation of antibody responses against the SARS-CoV-2 Spike protein and commensal microbiota via molecular mimicry.

The commensal microflora provides a repertoire of antigens that illicit mucosal antibodies. In some cases, these antibodies can cross-react with host proteins, inducing autoimmunity, or with other microbial antigens. We demonstrate that the oral microbiota can induce salivary anti-SARS-CoV-2 Spike IgG antibodies via molecular mimicry. Anti-Spike IgG antibodies in the saliva correlated with enhanced abundance of Streptococcus salivarius 1 month after anti-SARS-CoV-2 vaccination. Several human commensal bacteria, including S. salivarius, were recognized by SARS-CoV-2-neutralizing monoclonal antibodies and induced cross-reactive anti-Spike antibodies in mice, facilitating SARS-CoV-2 clearance. A specific S. salivarius protein, RSSL-01370, contains regions with homology to the Spike receptor-binding domain, and immunization of mice with RSSL-01370 elicited anti-Spike IgG antibodies in the serum. Additionally, oral S. salivarius supplementation enhanced salivary anti-Spike antibodies in vaccinated individuals. Altogether, these data show that distinct species of the human microbiota can express molecular mimics of SARS-CoV-2 Spike protein, potentially enhancing protective immunity.

Chimeric autoantibody receptor T cells deplete NMDA receptor-specific B cells.

Anti-NMDA receptor (NMDAR) autoantibodies cause NMDAR encephalitis, the most common autoimmune encephalitis, leading to psychosis, seizures, and autonomic dysfunction. Current treatments comprise broad immunosuppression or non-selective antibody removal. We developed NMDAR-specific chimeric autoantibody receptor (NMDAR-CAAR) T cells to selectively eliminate anti-NMDAR B cells and disease-causing autoantibodies. NMDAR-CAARs consist of an extracellular multi-subunit NMDAR autoantigen fused to intracellular 4-1BB/CD3ζ domains. NMDAR-CAAR T cells recognize a large panel of human patient-derived autoantibodies, release effector molecules, proliferate, and selectively kill antigen-specific target cell lines even in the presence of high autoantibody concentrations. In a passive transfer mouse model, NMDAR-CAAR T cells led to depletion of an anti-NMDAR B cell line and sustained reduction of autoantibody levels without notable off-target toxicity. Treatment of patients may reduce side effects, prevent relapses, and improve long-term prognosis. Our preclinical work paves the way for CAAR T cell phase I/II trials in NMDAR encephalitis and further autoantibody-mediated diseases.

Impaired functional connectivity of the hippocampus in translational murine models of NMDA-receptor antibody associated neuropsychiatric pathology.

Decreased hippocampal connectivity and disruption of functional networks are established resting-state functional MRI (rs-fMRI) features that are associated with neuropsychiatric symptom severity in human anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. However, the underlying pathophysiology of NMDAR encephalitis remains poorly understood. Application of patient-derived monoclonal antibodies against the NR1 (GluN1) subunit of the NMDAR now allows for the translational investigation of functional connectivity in experimental murine NMDAR antibody disease models with neurodevelopmental disorders. Using rs-fMRI, we studied functional connectivity alterations in (1) adult C57BL/6 J mice that were intrathecally injected with a recombinant human NR1 antibody over 14 days (n = 10) and in (2) a newly established mouse model with in utero exposure to a human recombinant NR1 antibody (NR1-offspring) at the age of (2a) 8 weeks (n = 15) and (2b) 10 months (n = 14). Adult NR1-antibody injected mice showed impaired functional connectivity within the left hippocampus compared to controls, resembling impaired connectivity patterns observed in human NMDAR encephalitis patients. Similarly, NR1-offspring showed significantly reduced functional connectivity in the hippocampus after 8 weeks, and impaired connectivity in the hippocampus was likewise observed in NR1-offspring at the age of 10 months. We successfully reproduced functional connectivity changes within the hippocampus in different experimental murine systems that were previously observed in human NMDAR encephalitis patients. Translational application of this method within a combined imaging and histopathological framework will allow future experimental studies to identify the underlying biological mechanisms and may eventually facilitate non-invasive monitoring of disease activity and treatment responses in autoimmune encephalitis.

Synapsin autoantibodies during pregnancy are associated with fetal abnormalities.

Anti-neuronal autoantibodies can be transplacentally transferred during pregnancy and may cause detrimental effects on fetal development. It is unclear whether autoantibodies against synapsin-I, one of the most abundant synaptic proteins, are associated with developmental abnormalities in humans. We recruited a cohort of 263 pregnant women and detected serum synapsin-I IgG autoantibodies in 13.3% using cell-based assays. Seropositivity was strongly associated with abnormalities of fetal development including structural defects, intrauterine growth retardation, amniotic fluid disorders and neuropsychiatric developmental diseases in previous children (odds ratios of 3-6.5). Autoantibodies reached the fetal circulation and were mainly of IgG1/IgG3 subclasses. They bound to conformational and linear synapsin-I epitopes, five distinct epitopes were identified using peptide microarrays. The findings indicate that synapsin-I autoantibodies may be clinically useful biomarkers or even directly participate in the disease process of neurodevelopmental disorders, thus being potentially amenable to antibody-targeting interventional strategies in the future.

Human NMDAR autoantibodies disrupt excitatory-inhibitory balance, leading to hippocampal network hypersynchrony.

Anti-NMDA receptor autoantibodies (NMDAR-Abs) in patients with NMDAR encephalitis cause severe disease symptoms resembling psychosis and cause cognitive dysfunction. After passive transfer of patients' cerebrospinal fluid or human monoclonal anti-GluN1-autoantibodies in mice, we find a disrupted excitatory-inhibitory balance resulting from CA1 neuronal hypoexcitability, reduced AMPA receptor (AMPAR) signaling, and faster synaptic inhibition in acute hippocampal slices. Functional alterations are also reflected in widespread remodeling of the hippocampal proteome, including changes in glutamatergic and GABAergic neurotransmission. NMDAR-Abs amplify network γ oscillations and disrupt θ-γ coupling. A data-informed network model reveals that lower AMPAR strength and faster GABAA receptor current kinetics chiefly account for these abnormal oscillations. As predicted in silico and evidenced ex vivo, positive allosteric modulation of AMPARs alleviates aberrant γ activity, reinforcing the causative effects of the excitatory-inhibitory imbalance. Collectively, NMDAR-Ab-induced aberrant synaptic, cellular, and network dynamics provide conceptual insights into NMDAR-Ab-mediated pathomechanisms and reveal promising therapeutic targets that merit future in vivo validation.

Pediatric de novo movement disorders and ataxia in the context of SARS-CoV-2.

OBJECTIVE: In the fourth year of the COVID-19 pandemic, mortality rates decreased, but the risk of neuropsychiatric disorders remained the same, with a prevalence of 3.8% of pediatric cases, including movement disorders (MD) and ataxia. METHODS: In this study, we report on a 10-year-old girl with hemichorea after SARS-CoV-2 infection and immunostained murine brain with patient CSF to identify intrathecal antibodies. Additionally, we conducted a scoping review of children with MD and ataxia after SARS-CoV-2 infection. RESULTS: We detected antibodies in the patient's CSF binding unknown antigens in murine basal ganglia. The child received immunosuppression and recovered completely. In a scoping review, we identified further 32 children with de novo MD or ataxia after COVID-19. While in a minority of cases, MD or ataxia were a symptom of known clinical entities (e.g. ADEM, Sydenham's chorea), in most children, the etiology was suspected to be of autoimmune origin without further assigned diagnosis. (i) Children either presented with ataxia (79%), but different from the well-known postinfectious acute cerebellar ataxia (older age, less favorable outcome, or (ii) had hypo-/hyperkinetic MD (21%), which were choreatic in most cases. Besides 14% of spontaneous recovery, immunosuppression was necessary in 79%. Approximately one third of children only partially recovered. CONCLUSIONS: Infection with SARS-CoV-2 can trigger de novo MD in children. Most patients showed COVID-19-associated-ataxia and fewer-chorea. Our data suggest that patients benefit from immunosuppression, especially steroids. Despite treatment, one third of patients recovered only partially, which makes up an increasing cohort with neurological sequelae.

Preclinical safety and efficacy of a therapeutic antibody that targets SARS-CoV-2 at the sotrovimab face but is escaped by Omicron.

The recurrent emerging of novel viral variants of concern (VOCs) with evasion of preexisting antibody immunity upholds severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) case numbers and maintains a persistent demand for updated therapies. We selected the patient-derived antibody CV38-142 based on its potency and breadth against the VOCs Alpha, Beta, Gamma, and Delta for preclinical development into a therapeutic. CV38-142 showed in vivo efficacy in a Syrian hamster VOC infection model after post-exposure and therapeutic application and revealed a favorable safety profile in a human protein library screen and tissue cross-reactivity study. Although CV38-142 targets the same viral surface as sotrovimab, which maintains activity against Omicron, CV38-142 did not neutralize the Omicron lineages BA.1 and BA.2. These results highlight the contingencies of developing antibody therapeutics in the context of antigenic drift and reinforce the need to develop broadly neutralizing variant-proof antibodies against SARS-CoV-2.

Maternal synapsin autoantibodies are associated with neurodevelopmental delay.

Maternal autoantibodies can be transmitted diaplacentally, with potentially deleterious effects on neurodevelopment. Synapsin 1 (SYN1) is a neuronal protein that is important for synaptic communication and neuronal plasticity. While monoallelic loss of function (LoF) variants in the SYN1 gene result in X-linked intellectual disability (ID), learning disabilities, epilepsy, behavioral problems, and macrocephaly, the effect of SYN1 autoantibodies on neurodevelopment remains unclear. We recruited a clinical cohort of 208 mothers and their children with neurologic abnormalities and analyzed the role of maternal SYN1 autoantibodies. We identified seropositivity in 9.6% of mothers, and seropositivity was associated with an increased risk for ID and behavioral problems. Furthermore, children more frequently had epilepsy, macrocephaly, and developmental delay, in line with the SYN1 LoF phenotype. Whether SYN1 autoantibodies have a direct pathogenic effect on neurodevelopment or serve as biomarkers requires functional experiments.

Hodgkin Lymphoma Cell Lines and Tissues Express mGluR5: A Potential Link to Ophelia Syndrome and Paraneoplastic Neurological Disease.

Ophelia syndrome is characterized by the coincidence of severe neuropsychiatric symptoms, classical Hodgkin lymphoma, and the presence of antibodies to the metabotropic glutamate 5 receptor (mGluR5). Little is known about the pathogenetic link between these symptoms and the role that anti-mGluR5-antibodies play. We investigated lymphoma tissue from patients with Ophelia syndrome and with isolated classical Hodgkin lymphoma by quantitative immunocytochemistry for mGluR5-expression. Further, we studied the L-1236, L-428, L-540, SUP-HD1, KM-H2, and HDLM-2 classical Hodgkin lymphoma cell lines by FACS and Western blot for mGluR5-expression, and by transcriptome analysis. mGluR5 surface expression differed significantly in terms of receptor density, distribution pattern, and percentage of positive cells. The highest expression levels were found in the L-1236 line. RNA-sequencing revealed more than 800 genes that were higher expressed in the L-1236 line in comparison to the other classical Hodgkin lymphoma cell lines. High mGluR5-expression was associated with upregulation of PI3K/AKT and MAPK pathways and of downstream targets (e.g., EGR1) known to be involved in classical Hodgkin lymphoma progression. Finally, mGluR5 expression was increased in the classical Hodgkin lymphoma-tissue of our Ophelia syndrome patient in contrast to five classical Hodgkin lymphoma-patients without autoimmune encephalitis. Given the association of encephalitis and classical Hodgkin lymphoma in Ophelia syndrome, it is possible that mGluR5-expression in classical Hodgkin lymphoma cells not only drives tumor progression but also triggers anti-mGluR5 encephalitis even before classical Hodgkin lymphoma becomes manifest.

Brain blood vessel autoantibodies in patients with NMDA and GABAA receptor encephalitis: identification of unconventional Myosin-X as target antigen.

Introduction: The antibody repertoire from CSF-derived antibody-secreting cells and memory B-cells in patients with encephalitis contains a considerable number of antibodies that do not target the disease-defining autoantigen such as the GABA or NMDA receptors. This study focuses on the functional relevance of autoantibodies to brain blood vessels in patients with GABAA and NMDA receptor encephalitis. Methods: We tested 149 human monoclonal IgG antibodies from the cerebrospinal fluid of six patients with different forms of autoimmune encephalitis on murine brain sections for reactivity to blood vessels using immunohistochemistry. Positive candidates were tested for reactivity with purified brain blood vessels, effects on transendothelial electrical resistance (TEER), and expression of tight junction proteins as well as gene regulation using human brain microvascular endothelial hCMEC/D3 cells as in vitro blood-brain barrier model. One blood-vessel reactive antibody was infused intrathecally by pump injection in mice to study in vivo binding and effects on tight junction proteins such as Occludin. Target protein identification was addressed using transfected HEK293 cells. Results: Six antibodies reacted with brain blood vessels, three were from the same patient with GABAAR encephalitis, and the other three were from different patients with NMDAR encephalitis. One antibody from an NMDAR encephalitis patient, mAb 011-138, also reacted with cerebellar Purkinje cells. In this case, treatment of hCMEC/D3 cells resulted in decreased TEER, reduced Occludin expression, and mRNA levels. Functional relevance in vivo was confirmed as Occludin downregulation was observed in mAb 011-138-infused animals. Unconventional Myosin-X was identified as a novel autoimmune target for this antibody. Discussion: We conclude that autoantibodies to blood vessels occur in autoimmune encephalitis patients and might contribute to a disruption of the blood-brain barrier thereby suggesting a potential pathophysiological relevance of these antibodies.

Differential Modes of Action of α1- and α1γ2-Autoantibodies Derived from Patients with GABAAR Encephalitis.

Autoantibodies against central nervous system proteins are increasingly being recognized in association with neurologic disorders. Although a growing number of neural autoantibodies have been identified, a causal link between specific autoantibodies and disease symptoms remains unclear, as most studies use patient-derived CSF-containing mixtures of autoantibodies. This raises questions concerning mechanism of action and which autoantibodies truly contribute to disease progression. To address this issue, monoclonal autoantibodies were isolated from a young girl with a range of neurologic symptoms, some of which reacted with specific GABAA receptor (GABAAR) subunits, α1-subunit and α1γ2-subunit, which in this study we have characterized in detail using a combination of cellular imaging and electrophysiological techniques. These studies in neurons from wild-type mice (C57BL/6J; RRID:IMSR_JAX:000664) of mixed-sex revealed that the α1 and α1γ2 subunit-specific antibodies have differential effects on the GABAA receptor. Namely, the α1-antibody was found to directly affect GABAA receptor function on a short time scale that diminished GABA currents, leading to increased network excitability. On longer time scales those antibodies also triggered a redistribution of the GABAA receptor away from synapses. In contrast, the α1γ2-antibody had no direct effect on GABAA receptor function and could possibly mediate its effect through other actors of the immune system. Taken together, these data highlight the complexity underlying autoimmune disorders and show that antibodies can exert their effect through many mechanisms within the same disease.

Structural mechanisms of GABAA receptor autoimmune encephalitis.

Autoantibodies targeting neuronal membrane proteins can cause encephalitis, seizures, and severe behavioral abnormalities. While antibodies for several neuronal targets have been identified, structural details on how they regulate function are unknown. Here we determined cryo-electron microscopy structures of antibodies derived from an encephalitis patient bound to the γ-aminobutyric acid type A (GABAA) receptor. These antibodies induced severe encephalitis by directly inhibiting GABAA function, resulting in nervous-system hyperexcitability. The structures reveal mechanisms of GABAA inhibition and pathology. One antibody directly competes with a neurotransmitter and locks the receptor in a resting-like state. The second antibody targets the subunit interface involved in binding benzodiazepines and antagonizes diazepam potentiation. We identify key residues in these antibodies involved in specificity and affinity and confirm structure-based hypotheses for functional effects using electrophysiology. Together these studies define mechanisms of direct functional antagonism of neurotransmission underlying autoimmune encephalitis in a human patient.

Key benefits of dexamethasone and antibody treatment in COVID-19 hamster models revealed by single-cell transcriptomics.

For coronavirus disease 2019 (COVID-19), effective and well-understood treatment options are still scarce. Since vaccine efficacy is challenged by novel variants, short-lasting immunity, and vaccine hesitancy, understanding and optimizing therapeutic options remains essential. We aimed at better understanding the effects of two standard-of-care drugs, dexamethasone and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies, on infection and host responses. By using two COVID-19 hamster models, pulmonary immune responses were analyzed to characterize effects of single or combinatorial treatments. Pulmonary viral burden was reduced by anti-SARS-CoV-2 antibody treatment and unaltered or increased by dexamethasone alone. Dexamethasone exhibited strong anti-inflammatory effects and prevented fulminant disease in a severe disease model. Combination therapy showed additive benefits with both anti-viral and anti-inflammatory potency. Bulk and single-cell transcriptomic analyses confirmed dampened inflammatory cell recruitment into lungs upon dexamethasone treatment and identified a specifically responsive subpopulation of neutrophils, thereby indicating a potential mechanism of action. Our analyses confirm the anti-inflammatory properties of dexamethasone and suggest possible mechanisms, validate anti-viral effects of anti-SARS-CoV-2 antibody treatment, and reveal synergistic effects of a combination therapy, thus informing more effective COVID-19 therapies.

Patient-Derived Anti-NMDAR Antibody Disinhibits Cortical Neuronal Networks through Dysfunction of Inhibitory Neuron Output.

Anti-NMDA receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder associated with autoantibodies against NMDARs, which cause a variety of symptoms from prominent psychiatric and cognitive manifestations to seizures and autonomic instability. Previous studies mainly focused on hippocampal effects of these autoantibodies, helping to explain mechanistic causes for cognitive impairment. However, antibodies' effects on higher cortical network function, where they could contribute to psychosis and/or seizures, have not been explored in detail until now. Here, we employed a patient-derived monoclonal antibody targeting the NR1 subunit of NMDAR and tested its effects on in vitro cultures of rodent cortical neurons, using imaging and electrophysiological techniques. We report that this hNR1 antibody drives cortical networks to a hyperexcitable state and disrupts mechanisms stabilizing network activity such as Npas4 signaling. Network hyperactivity is in part a result of a reduced synaptic output of inhibitory neurons, as indicated by a decreased inhibitory drive and levels of presynaptic inhibitory proteins, specifically in inhibitory-to-excitatory neuron synapses. Importantly, on a single-cell level hNR1 antibody selectively impairs NMDAR-mediated currents and synaptic transmission of cortical inhibitory neurons, yet has no effect on excitatory neurons, which contrasts with its effects on hippocampal neurons. Together, these findings provide a novel, cortex-specific mechanism of antibody-induced neuronal hyperexcitability, highlighting regional specificity underlying the pathology of autoimmune encephalitis.SIGNIFICANCE STATEMENT It is increasingly appreciated that the inadvertent activation of the immune system within CNS can underlie pathogenesis of neuropsychiatric disorders. Although the exact mechanisms remain elusive, autoantibodies derived from patients with autoimmune encephalitis pose a unique tool to study pathogenesis of neuropsychiatric states. Our analysis reveals that autoantibody against the NMDA receptor (NMDAR) has a distinct mechanism of action in the cortex, where it impairs function of inhibitory neurons leading to increased cortical network excitability, in contrast to previously described hippocampal synaptic mechanisms of information encoding, highlighting brain regional specificity. Notably, similar mechanism of NMDAR-mediated inhibitory hypofunction leading to cortical disinhibition has been suggested to underlie pathology of schizophrenia, hence our data provide new evidence for common mechanisms underlying neuropsychiatric disorders.

SARS-CoV-2 Beta variant infection elicits potent lineage-specific and cross-reactive antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Beta variant of concern (VOC) resists neutralization by major classes of antibodies from COVID-19 patients and vaccinated individuals. In this study, serum of Beta-infected patients revealed reduced cross-neutralization of wild-type virus. From these patients, we isolated Beta-specific and cross-reactive receptor-binding domain (RBD) antibodies. The Beta-specificity results from recruitment of VOC-specific clonotypes and accommodation of mutations present in Beta and Omicron into a major antibody class that is normally sensitive to these mutations. The Beta-elicited cross-reactive antibodies share genetic and structural features with wild type-elicited antibodies, including a public VH1-58 clonotype that targets the RBD ridge. These findings advance our understanding of the antibody response to SARS-CoV-2 shaped by antigenic drift, with implications for design of next-generation vaccines and therapeutics.

Encephalitis patient-derived monoclonal GABAA receptor antibodies cause epileptic seizures.

Autoantibodies targeting the GABAA receptor (GABAAR) hallmark an autoimmune encephalitis presenting with frequent seizures and psychomotor abnormalities. Their pathogenic role is still not well-defined, given the common overlap with further autoantibodies and the lack of patient-derived mAbs. Five GABAAR mAbs from cerebrospinal fluid cells bound to various epitopes involving the α1 and γ2 receptor subunits, with variable binding strength and partial competition. mAbs selectively reduced GABAergic currents in neuronal cultures without causing receptor internalization. Cerebroventricular infusion of GABAAR mAbs and Fab fragments into rodents induced a severe phenotype with seizures and increased mortality, reminiscent of encephalitis patients' symptoms. Our results demonstrate direct pathogenicity of autoantibodies on GABAARs independent of Fc-mediated effector functions and provide an animal model for GABAAR encephalitis. They further provide the scientific rationale for clinical treatments using antibody depletion and can serve as tools for the development of antibody-selective immunotherapies.

Multimodal electrophysiological analyses reveal that reduced synaptic excitatory neurotransmission underlies seizures in a model of NMDAR antibody-mediated encephalitis.

Seizures are a prominent feature in N-Methyl-D-Aspartate receptor antibody (NMDAR antibody) encephalitis, a distinct neuro-immunological disorder in which specific human autoantibodies bind and crosslink the surface of NMDAR proteins thereby causing internalization and a state of NMDAR hypofunction. To further understand ictogenesis in this disorder, and to test a potential treatment compound, we developed an NMDAR antibody mediated rat seizure model that displays spontaneous epileptiform activity in vivo and in vitro. Using a combination of electrophysiological and dynamic causal modelling techniques we show that, contrary to expectation, reduction of synaptic excitatory, but not inhibitory, neurotransmission underlies the ictal events through alterations in the dynamical behaviour of microcircuits in brain tissue. Moreover, in vitro application of a neurosteroid, pregnenolone sulphate, that upregulates NMDARs, reduced established ictal activity. This proof-of-concept study highlights the complexity of circuit disturbances that may lead to seizures and the potential use of receptor-specific treatments in antibody-mediated seizures and epilepsy.