18 research outputs found
Brain antibody sequence evaluation (BASE): an easy-to-use software for complete data analysis in single cell immunoglobulin cloning
Background:
Repertoire analysis of patient-derived recombinant monoclonal antibodies is an important tool to study the role of B cells in autoimmune diseases of the human brain and beyond. Current protocols for generation of patient-derived recombinant monoclonal antibody libraries are time-consuming and contain repetitive steps, some of which can be assisted with the help of software automation.
Results:
We developed BASE, an easy-to-use software for complete data analysis in single cell immunoglobulin cloning. BASE consists of two modules: aBASE for immunological annotations and cloning primer lookup, and cBASE for plasmid sequence identity confirmation before expression. Comparing automated BASE analysis with manual analysis we confirmed the validity of BASE output: identity between manual and automated aBASE analysis was 100% for all outputs, except for immunoglobulin isotype determination. In this case, aBASE yielded correct results in 96% of cases, whereas 4% of cases required manual confirmation. cBASE automatically concluded expression recommendations in 89.8% of cases, 91.8% of which were identical to manually derived results and none of them were false-positive.
Conclusions:
BASE offers an easy-to-use software solution suitable for complete Ig sequence data analysis and tracking during recombinant mAb cloning from single cells. Plasmid sequence identity confirmation by cBASE offers functionality not provided by existing software solutions in the field and will help to reduce time-consuming steps of the monoclonal antibody generation workflow. BASE can be installed locally or accessed online at Code Ocean
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
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
Human gestational Nâmethylâdâaspartate receptor autoantibodies impair neonatal murine brain function
Objective: Maternal autoantibodies are a risk factor for impaired brain development in offspring. Antibodies (ABs) against the NR1 (GluN1) subunit of the N-methyl-d-aspartate receptor (NMDAR) are among the most frequently diagnosed anti-neuronal surface ABs, yet little is known about effects on fetal development during pregnancy.
Methods: We established a murine model of in utero exposure to human recombinant NR1 and isotype-matched nonreactive control ABs. Pregnant C57BL/6J mice were intraperitoneally injected on embryonic days 13 and 17 each with 240ÎŒg of human monoclonal ABs. Offspring were investigated for acute and chronic effects on NMDAR function, brain development, and behavior.
Results: Transferred NR1 ABs enriched in the fetus and bound to synaptic structures in the fetal brain. Density of NMDAR was considerably reduced (up to -49.2%) and electrophysiological properties were altered, reflected by decreased amplitudes of spontaneous excitatory postsynaptic currents in young neonates (-34.4%). NR1 AB-treated animals displayed increased early postnatal mortality (+27.2%), impaired neurodevelopmental reflexes, altered blood pH, and reduced bodyweight. During adolescence and adulthood, animals showed hyperactivity (+27.8% median activity over 14 days), lower anxiety, and impaired sensorimotor gating. NR1 ABs caused long-lasting neuropathological effects also in aged mice (10 months), such as reduced volumes of cerebellum, midbrain, and brainstem.
Interpretation: The data collectively support a model in which asymptomatic mothers can harbor low-level pathogenic human NR1 ABs that are diaplacentally transferred, causing neurotoxic effects on neonatal development. Thus, AB-mediated network changes may represent a potentially treatable neurodevelopmental congenital brain disorder contributing to lifelong neuropsychiatric morbidity in affected children
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
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
Repertoire analysis of monoclonal antibodies in the cerebrospinal fluid from patients with anti-NMDA-Receptor-Encephalitis
Die Anti-N-Methyl-D-Aspartat-Rezeptor(NMDAR)-Enzephalitis ist die hÀufigste
Form autoimmun vermittelter HirnentzĂŒndungen. Die PatientInnen entwickeln
typischerweise zunÀchst psychotische VerÀnderungen wie inhaltliche
Denkstörungen und Halluzinationen und in der Folge neurologische Symptome, wie
Bewusstseinsstörungen, Dyskinesien, epileptische AnfÀlle, autonome
Dysregulationen und zentrale Hypoventilation. Der Nachweis von Antikörpern
gegen die NR1-Untereinheit des NMDAR im Liquor gilt als wichtigstes
Diagnosekriterium. Bisherige Arbeiten mit extrahiertem Gesamt-Immunglobulin
aus Liquor und Serum von PatientInnen zeigten Störungen der synaptischen
Funktion in vitro und in vivo. Der Einfluss weiterer möglicher Autoantikörper
konnte dabei nicht ausgeschlossen werden und der Beweis fĂŒr die PathogenitĂ€t
NR1-spezifischer Antikörper war bisher ausstehend. Dazu generierten wir
monoklonale rekombinante humane Antikörper aus Liquorproben von acht
Patientinnen mit NMDAR-Enzephalitis. Wir isolierten einzelne
GedÀchtnis-B-Zellen sowie Plasmazellen mittels Durchflusszytometrie. Die
variablen Regionen der fĂŒr die schweren und leichten Immunglobulinketten
kodierenden Genabschnitte amplifizierten mittels Polymerase-Kettenreaktion.
Diese klonierten wir in Vektoren, die den konstanten Genabschnitt humaner
Antikörper enthalten. Mit dem jeweiligen Vektorenpaar aus schwerer und
leichter Immunglobulinkette co-transfizierten wir menschliche embryonale
Nierenzellen (HEK-Zellen) und gewannen aus dem Kulturmedium die monoklonalen
Antikörper. Sechs Prozent der generierten rekombinanten humanen Antikörper
zeigten eine ReaktivitĂ€t auf NR1-transfizierten HEK-Zellen, ebenso das fĂŒr
NR1-spezifische Antikörper typische FÀrbemuster auf hippocampalen Neuronen und
auf MÀusehirnschnitten. Alle NR1-spezifischen Antikörper waren vom IgG-Isotyp,
sie lieĂen sich aus Plasmazellen sowie GedĂ€chtnis-B-Zellen isolieren. Letztere
könnten als Ausgangspunkt möglicher spÀterer Rezidive klinisch relevant sein.
Einige der NR1-spezifischen Antikörper zeigten eine klonale Expansion mit 100
Prozent identischen Klonen und wiesen wenige somatische Hypermutationen auf,
was auf eine kĂŒrzlich stattgefundene periphere Immunreaktion hindeutet. Wir
fanden zudem drei völlig unmutierte NR1-spezifische Antikörper (ânaturally
occuring antibodiesâ), was bei diesen Patientinnen eine unvollstĂ€ndige
Immuntoleranz gegenĂŒber NR1 nahelegt. Auf hippocampalen Neuronen fĂŒhrte die
Inkubation mit monoklonalen NR1-spezifischen Antikörpern zu einer Senkung der
NMDAR-Dichte sowie zu einer Reduktion der NMDAR-spezifischen Ströme. Nach
intravenösen Injektionen in MÀuse lieà sich eine Anreicherung der Antikörper
im Hippocampus und Kleinhirn nachweisen. FĂŒr ĂŒber 95 Prozent der nicht
NR1-bindenden Antikörper zeigte sich eine spezifische ReaktivitÀt auf
MÀusehirnschnitten, unter anderem an neuronalen OberflÀchen, an Gliazellen und
an Endothel. Mit diesen Daten konnten wir nachweisen, dass monoklonale
NR1-spezifische Antikörper allein neurotoxisch sind. Damit ist jeder Nachweis
dieser Autoantikörper als ein Risikofaktor fĂŒr neuropsychiatrische Symptome
anzusehen. In zukĂŒnftigen Arbeiten gilt es zu klĂ€ren, ob das Vorhandensein
verschiedener NR1-spezifischer Antikörper prognostisch relevant ist und ob
weitere Autoantikörper zur Pathophysiologie beitragen.The anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most common
form of autoimmune mediated brain inflammation. Typically patients initially
develop psychotic changes such as substantive thought disorders and
hallucinations, followed by neurological symptoms like impaired consciousness,
dyskinesia, seizures, autonomic dysregulations and central hypoventilation.
The detection of antibodies against the NR1 subunit of the NMDAR in the
cerebrospinal fluid (CSF) is the most important diagnostic criterion. Previous
works with extracted whole immunoglobulins from patientsâ CSF and serum have
shown synaptic dysfunction in vitro and in vivo. Thereby an influence of
further possible autoantibodies could not be eliminated and the proof of
pathogenicity of NR1-specific antibodies was pending. Therefore we generated
monoclonal recombinant human antibodies from CSF samples from eight patients
with NMDAR encephalitis. We isolated single memory B cells and plasma cells
via flow cytometry. The variable gene regions coding the heavy and light
immunoglobulin chains were amplified by polymerase chain reaction. These were
cloned into vectors, containing the constant gene sequence of human
antibodies. We co-transfected human embryonic kidney (HEK) cells with the
respective pair of heavy and light immunoglobulin chains and harvested
monoclonal antibodies from the culture medium. Six percent of the generated
monoclonal recombinant human antibodies showed reactivity to NR1-transfected
HEK cells, likewise the typical NR1-specific staining pattern on hippocampal
neurons and mouse brain sections. All NR1-specific antibodies were from the
IgG isotype and have been isolated from plasma cells as well as memory B
cells. Last-mentioned ones might be clinically relevant as possible origin of
future relapses. Several of the NR1-specific antibodies showed clonal
expansion with 100 percent identical clones and few somatic hypermutations,
indicating a recent peripheral immune reaction. Furthermore we found three
completely unmutated NR1-specific antibodies (ânaturally occurring
antibodiesâ), suggesting incomplete immune tolerance against NR1 in these
patients. Incubating hippocampal neurons with monoclonal NR1-specific
antibodies caused a reduction of the NMDAR density and NMDAR-specific
currents. After intravenous injections into mice, antibody enrichment in the
hippocampus and cerebellum was detected. More than 95 percent of the non-
NR1-binding antibodies showed specific reactivity on mouse brain sections,
amongst others to neuronal surfaces, glia cells and endothelium. With these
data we proved that monoclonal NR1-specific antibodies alone are neurotoxic.
Therewith, any detection of these autoantibodies needs to be considered as a
risk factor for neuro-psychiatric symptoms. Future work will clarify whether
the presence of different NR1-specific antibodies is of prognostic relevance
and whether further autoantibodies contribute to the pathophysiology
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