Memory loss and aberrant neurogenesis in mice exposed to patient anti-N-methyl-d-aspartate receptor antibodies.

OBJECTIVE: Anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis results in chronic epilepsy and permanent cognitive impairment. One of the possible causes of cognitive impairment in anti-NMDAR could be aberrant neurogenesis, an established contributor to memory loss in idiopathic drug-resistant epilepsy. We developed a mouse model of anti-NMDAR encephalitis and showed that mice exposed to patient anti-NMDAR antibodies for 2 weeks developed seizures and memory loss. In the present study, we assessed the delayed effects of patient-derived antibodies on cognitive phenotype and examined the corresponding changes in hippocampal neurogenesis. METHODS: Monoclonal anti-NMDAR antibodies or control antibodies were continuously infused into the lateral ventricle of male C56BL/6 J mice (8-12 weeks) via osmotic minipumps for 2 weeks. The motor and anxiety phenotypes were assessed using the open field paradigm, and hippocampal memory and learning were assessed using the object location, Y maze, and Barnes maze paradigms during weeks 1 and 3-4 of antibody washout. The numbers of newly matured granule neurons (Prox-1+) and immature progenitor cells (DCX+) as well as their spatial distribution within the hippocampus were assessed at these time points. Bromodeoxyuridine (BrdU: 50 mg/kg, i.p., daily) was injected on days 2-12 of the infusion, and proliferating cell immunoreactivity was compared in antibody-treated mice and control mice at week 3 of the washout. RESULTS: Mice infused with anti-NMDAR antibodies demonstrated spatial memory impairment during week 1 of antibody washout (p = 0.02, t-test; n = 9-11). Histological analysis of hippocampal sections from these mice revealed an increased ectopic displacement of Prox-1+ cells in the dentate hilus compared to the control-antibody-treated mice (p = 0.01; t-test). Mice exposed to anti-NMDAR antibodies also had an impairment of spatial memory and learning during weeks 3-4 of antibody washout (object location: p = 0.009; t-test; Y maze: p = 0.006, t-test; Barnes maze: p = 0.008, ANOVA; n = 8-10). These mice showed increased ratios of the low proliferating (bright) to fast proliferating (faint) BrdU+ cell counts and decreased number of DCX+ cells in the hippocampal dentate gyrus (p = 0.006 and p = 0.04, respectively; t-tests) suggesting ectopic migration and delayed cell proliferation. SIGNIFICANCE: These findings suggest that memory and learning impairments induced by patient anti-NMDAR antibodies are sustained upon removal of antibodies and are accompanied by aberrant hippocampal neurogenesis. Interventions directed at the manipulation of neuronal plasticity in patients with encephalitis and cognitive loss may be protective and therapeutically relevant.

MyD88-mediated signaling is critical for the generation of seizure responses and cognitive impairment in a model of anti-N-methyl-D-aspartate receptor encephalitis.

OBJECTIVE: We previously demonstrated that interleukin-1 receptor-mediated immune activation contributes to seizure severity and memory loss in anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. In the present study, we assessed the role of the myeloid differentiation primary response gene 88 (MyD88), an adaptor protein in Toll-like receptor signaling, in the key phenotypic characteristics of anti-NMDAR encephalitis. METHODS: Monoclonal anti-NMDAR antibodies or control antibodies were infused into the lateral ventricle of MyD88 knockout mice (MyD88-/-) and control C56BL/6J mice (wild type [WT]) via osmotic minipumps for 2 weeks. Seizure responses were measured by electroencephalography. Upon completion of the infusion, the motor, anxiety, and memory functions of the mice were assessed. Astrocytic (glial fibrillary acidic protein [GFAP]) and microglial (ionized calcium-binding adaptor molecule 1 [Iba-1]) activation and transcriptional activation for the principal inflammatory mediators involved in seizures were determined using immunohistochemistry and quantitative real-time polymerase chain reaction, respectively. RESULTS: As shown before, 80% of WT mice infused with anti-NMDAR antibodies (n = 10) developed seizures (median = 11, interquartile range [IQR] = 3-25 in 2 weeks). In contrast, only three of 14 MyD88-/- mice (21.4%) had seizures (0, IQR = 0-.25, p = .01). The WT mice treated with antibodies also developed memory loss in the novel object recognition test, whereas such memory deficits were not apparent in MyD88-/- mice treated with anti-NMDAR antibodies (p = .03) or control antibodies (p = .04). Furthermore, in contrast to the WT mice exposed to anti-NMDAR antibodies, the MyD88-/- mice had a significantly lower induction of chemokine (C-C motif) ligand 2 (CCL2) in the hippocampus (p = .0001, Sidak tests). There were no significant changes in the expression of GFAP and Iba-1 in the MyD88-/- mice treated with anti-NMDAR or control antibodies. SIGNIFICANCE: These findings suggest that MyD88-mediated signaling contributes to the seizure and memory phenotype in anti-NMDAR encephalitis and that CCL2 activation may participate in the expression of these features. The removal of MyD88 inflammation may be protective and therapeutically relevant.

Recombinant neutralizing secretory IgA antibodies for preventing mucosal acquisition and transmission of SARS-CoV-2.

Passive delivery of antibodies to mucosal sites may be a valuable adjunct to COVID-19 vaccination to prevent infection, treat viral carriage, or block transmission. Neutralizing monoclonal IgG antibodies are already approved for systemic delivery, and several clinical trials have been reported for delivery to mucosal sites where SARS-CoV-2 resides and replicates in early infection. However, secretory IgA may be preferred because the polymeric complex is adapted for the harsh, unstable external mucosal environment. Here, we investigated the feasibility of producing neutralizing monoclonal IgA antibodies against SARS-CoV-2. We engineered two class-switched mAbs that express well as monomeric and secretory IgA (SIgA) variants with high antigen-binding affinities and increased stability in mucosal secretions compared to their IgG counterparts. SIgAs had stronger virus neutralization activities than IgG mAbs and were protective against SARS-CoV-2 infection in an in vivo murine model. Furthermore, SIgA1 can be aerosolized for topical delivery using a mesh nebulizer. Our findings provide a persuasive case for developing recombinant SIgAs for mucosal application as a new tool in the fight against COVID-19.

A human monoclonal antibody binds within the poliovirus receptor-binding site to neutralize all three serotypes.

Global eradication of poliovirus remains elusive, and it is critical to develop next generation vaccines and antivirals. In support of this goal, we map the epitope of human monoclonal antibody 9H2 which is able to neutralize the three serotypes of poliovirus. Using cryo-EM we solve the near-atomic structures of 9H2 fragments (Fab) bound to capsids of poliovirus serotypes 1, 2, and 3. The Fab-virus complexes show that Fab interacts with the same binding mode for each serotype and at the same angle of interaction relative to the capsid surface. For each of the Fab-virus complexes, we find that the binding site overlaps with the poliovirus receptor (PVR) binding site and maps across and into a depression in the capsid called the canyon. No conformational changes to the capsid are induced by Fab binding for any complex. Competition binding experiments between 9H2 and PVR reveal that 9H2 impedes receptor binding. Thus, 9H2 outcompetes the receptor to neutralize poliovirus. The ability to neutralize all three serotypes, coupled with the critical importance of the conserved receptor binding site make 9H2 an attractive antiviral candidate for future development.

Impact of Simultaneous Circulating Tumor DNA and Tissue Genotyping in the Workup of Stage IV Lung Adenocarcinoma on Quality of Care in an Academic Community Medical Center.

PURPOSE: In patients with metastatic lung adenocarcinoma, evidence-based first-line treatment decisions require analysis of tumors for genomic alterations (GAs). Optimizing the genotyping paradigm may improve the delivery of precision oncology care. Actionable GAs can be identified by analyzing tumor tissue or circulating tumor DNA using liquid biopsy. Consensus guidelines for when to use liquid biopsy have not been established. We evaluated the routine use of liquid biopsy performed simultaneously with tissue testing in patients with newly diagnosed, stage IV lung adenocarcinoma. METHODS: We performed a retrospective study comparing patients who underwent tissue genotyping alone (standard biopsy group) with patients who had simultaneous liquid and tissue genotyping (combined biopsy group). We examined the time to reach a final diagnosis, the need for repeat biopsies, and diagnostic accuracy. RESULTS: Forty two patients in the combined biopsy group and 78 in the standard biopsy group met the inclusion criteria. The standard group had a mean time to diagnosis of 33.5 days, compared with 20.6 days in the combined group (P < .001 by two-tailed t-test). In the combined group, 14 patients did not have sufficient tissue for molecular analysis (30%); however, in 11 (79%) of these patients, liquid biopsy identified a GA that eliminated the need for a second tissue biopsy. In patients who completed both tests, each test found actionable GAs missed by the other. CONCLUSION: Performing liquid biopsy simultaneously with tissue genotyping is feasible in an academic community medical center. Potential advantages of simultaneous liquid and tissue biopsies include shorter time to obtain a definitive molecular diagnosis, reduced need for a repeat biopsy, and improved detection of actionable mutations, although a sequential strategy that saves costs by beginning with a liquid biopsy may be ideal.

Regulation of NMDA Receptor Signaling at Single Synapses by Human Anti-NMDA Receptor Antibodies.

The NMDA receptor (NMDAR) subunit GluN1 is critical for receptor function and plays a pivotal role in synaptic plasticity. Mounting evidence has shown that pathogenic autoantibody targeting of the GluN1 subunit of NMDARs, as in anti-NMDAR encephalitis, leads to altered NMDAR trafficking and synaptic localization. However, the underlying signaling pathways affected by antibodies targeting the NMDAR remain to be fully delineated. It remains unclear whether patient antibodies influence synaptic transmission via direct effects on NMDAR channel function. Here, we show using short-term incubation that GluN1 antibodies derived from patients with anti-NMDAR encephalitis label synapses in mature hippocampal primary neuron culture. Miniature spontaneous calcium transients (mSCaTs) mediated via NMDARs at synaptic spines are not altered in pathogenic GluN1 antibody exposed conditions. Unexpectedly, spine-based and cell-based analyses yielded distinct results. In addition, we show that calcium does not accumulate in neuronal spines following brief exposure to pathogenic GluN1 antibodies. Together, these findings show that pathogenic antibodies targeting NMDARs, under these specific conditions, do not alter synaptic calcium influx following neurotransmitter release. This represents a novel investigation of the molecular effects of anti-NMDAR antibodies associated with autoimmune encephalitis.

A Strategy to Detect Emerging Non-Delta SARS-CoV-2 Variants with a Monoclonal Antibody Specific for the N501 Spike Residue.

Efforts to control SARS-CoV-2 have been challenged by the emergence of variant strains that have important implications for clinical and epidemiological decision making. Four variants of concern (VOCs) have been designated by the Centers for Disease Control and Prevention (CDC), namely, B.1.617.2 (delta), B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma), although the last three have been downgraded to variants being monitored (VBMs). VOCs and VBMs have shown increased transmissibility and/or disease severity, resistance to convalescent SARS-CoV-2 immunity and antibody therapeutics, and the potential to evade diagnostic detection. Methods are needed for point-of-care (POC) testing to rapidly identify these variants, protect vulnerable populations, and improve surveillance. Antigen-detection rapid diagnostic tests (Ag-RDTs) are ideal for POC use, but Ag-RDTs that recognize specific variants have not yet been implemented. Here, we describe a mAb (2E8) that is specific for the SARS-CoV-2 spike protein N501 residue. The 2E8 mAb can distinguish the delta VOC from variants with the N501Y meta-signature, which is characterized by convergent mutations that contribute to increased virulence and evasion of host immunity. Among the N501Y-containing mutants formerly designated as VOCs (alpha, beta, and gamma), a previously described mAb, CB6, can distinguish beta from alpha and gamma. When used in a sandwich ELISA, these mAbs sort these important SARS-CoV-2 variants into three diagnostic categories, namely, (1) delta, (2) alpha or gamma, and (3) beta. As delta is currently the predominant variant globally, they will be useful for POC testing to identify N501Y meta-signature variants, protect individuals in high-risk settings, and help detect epidemiological shifts among SARS-CoV-2 variants.

Monoclonal Antibodies From Anti-NMDA Receptor Encephalitis Patient as a Tool to Study Autoimmune Seizures.

Anti-N-methyl-D-aspartate (NMDA) receptor encephalitis manifests with precipitous cognitive decline, abnormal movements, and severe seizures that can be challenging to control with conventional anti-seizure medications. We previously demonstrated that intracerebroventricular (i.c.v.) administration of cerebrospinal fluid from affected patients, or purified NMDA receptor antibodies from encephalitis patients to mice precipitated seizures, thereby confirming that antibodies are directly pathogenic for seizures. Although different repertoires of anti-NMDA receptor antibodies could contribute to the distinct clinical manifestations in encephalitis patients, the role of specific antibodies in the expression of seizure, motor, and cognitive phenotypes remains unclear. Using three different patient-derived monoclonal antibodies with distinct epitopes within the N-terminal domain (NTD) of the NMDA receptor, we characterized the seizure burden, motor activity and anxiety-related behavior in mice. We found that continuous administration of 5F5, 2G6 or 3C11 antibodies for 2 weeks precipitated seizures, as measured with continuous EEG using cortical screw electrodes. The seizure burden was comparable in all three antibody-treated groups. The seizures were accompanied by increased hippocampal C-C chemokine ligand 2 (CCL2) mRNA expression 3 days after antibody infusion had stopped. Antibodies did not affect the motor performance or anxiety scores in mice. These findings suggest that neuronal antibodies targeting different epitopes within the NMDA receptor may result in a similar seizure phenotype.

Seizures and memory impairment induced by patient-derived anti-N-methyl-D-aspartate receptor antibodies in mice are attenuated by anakinra, an interleukin-1 receptor antagonist.

OBJECTIVE: Neuroinflammation associated with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis may facilitate seizures. We previously showed that intraventricular administration of cerebrospinal fluid from patients with anti-NMDAR encephalitis to mice precipitates seizures, thereby confirming that antibodies are directly pathogenic. To determine whether interleukin (IL)-1-mediated inflammation exacerbates autoimmune seizures, we asked whether blocking the effects of IL-1 by anakinra, a selective IL-1 receptor antagonist, blunts antibody-induced seizures. METHODS: We infused C57BL/6 mice intraventricularly with purified serum IgG from patients with anti-NMDAR encephalitis or monoclonal anti-NMDAR IgG; subdural electroencephalogram was continuously recorded. After a 6-day interval, mice received anakinra (25 mg/kg sc, twice daily) or vehicle for 5 days. Following a 4-day washout period, we performed behavioral tests to assess motor function, anxiety, and memory, followed by hippocampus tissue analysis to assess astrocytic (glial fibrillary acidic protein [GFAP]) and microglial (ionized calcium-binding adapter molecule [Iba]-1) activation. RESULTS: Of 31 mice infused with purified patient NMDAR-IgG (n = 17) or monoclonal NMDAR-IgG (n = 14), 81% developed seizures. Median baseline daily seizure count during exposure to antibodies was 3.9; most seizures were electrographic. Median duration of seizures during the baseline was 82.5 s. Anakinra administration attenuated daily seizure frequency by 60% (p = .02). Anakinra reduced seizure duration; however, the effect was delayed and became apparent only after the cessation of treatment (p = .04). Anakinra improved novel object recognition in mice with antibody-induced seizures (p = .03) but did not alter other behaviors. Anakinra reduced the expression of GFAP and Iba-1 in the hippocampus of mice with seizures, indicating decreased astrocytic and microglial activation. SIGNIFICANCE: Our evidence supports a role for IL-1 in the pathogenesis of seizures in anti-NMDAR encephalitis. These data are consistent with therapeutic effects of anakinra in other severe autoimmune and inflammatory seizure syndromes. Targeting inflammation via blocking IL-1 receptor-mediated signaling may be promising for developing novel treatments for refractory autoimmune seizures.

Human IgA Monoclonal Antibodies That Neutralize Poliovirus, Produced by Hybridomas and Recombinant Expression.

Poliovirus (PV)-specific intestinal IgAs are important for cessation of PV shedding in the gastrointestinal tract following an acute infection with wild type or vaccine-derived PV strains. We sought to produce IgA monoclonal antibodies (mAbs) with PV neutralizing activity. We first performed de novo IgA discovery from primary human B cells using a hybridoma method that allows assessment of mAb binding and expression on the hybridoma surface: On-Cell mAb Screening (OCMS™). Six IgA1 mAbs were cloned by this method; three potently neutralized type 3 Sabin and wt PV strains. The hybridoma mAbs were heterogeneous, expressed in monomeric, dimeric, and aberrant forms. We also used recombinant methods to convert two high-potency anti-PV IgG mAbs into dimeric IgA1 and IgA2 mAbs. Isotype switching did not substantially change their neutralization activities. To purify the recombinant mAbs, Protein L binding was used, and one of the mAbs required a single amino acid substitution in its κ LC in order to enable protein L binding. Lastly, we used OCMS to assess IgA expression on the surface of hybridomas and transiently transfected, adherent cells. These studies have generated potent anti-PV IgA mAbs, for use in animal models, as well as additional tools for the discovery and production of human IgA mAbs.

Salmonella Typhimurium biofilm disruption by a human antibody that binds a pan-amyloid epitope on curli.

Bacterial biofilms, especially those associated with implanted medical devices, are difficult to eradicate. Curli amyloid fibers are important components of the biofilms formed by the Enterobacteriaceae family. Here, we show that a human monoclonal antibody with pan-amyloid-binding activity (mAb 3H3) can disrupt biofilms formed by Salmonella enterica serovar Typhimurium in vitro and in vivo. The antibody disrupts the biofilm structure, enhancing biofilm eradication by antibiotics and immune cells. In mice, 3H3 injections allow antibiotic-mediated clearance of catheter-associated S. Typhimurium biofilms. Thus, monoclonal antibodies that bind a pan-amyloid epitope have potential to prevent or eradicate bacterial biofilms.

Universal ELISA for quantification of D-antigen in inactivated poliovirus vaccines.

To address the biosafety and biosecurity concerns related to the manufacture of inactivated polio vaccine (IPV), several manufacturers started producing it from attenuated Sabin strains. Slight immunological differences between wild and attenuated strains create a challenge for testing IPV potency, which is defined as the content of protective D-antigen determined in an ELISA test. Some ELISA reagents selected for testing conventional IPV made from wild strains (cIPV) may not be suitable for testing Sabin IPV (sIPV). This paper describes an ELISA procedure using human monoclonal antibodies selected to capture equally well both wild and attenuated strains of poliovirus. A unique monoclonal antibody neutralizing all three serotypes of poliovirus was used as the detection antibody. The method was shown to detect only D-antigen of both conventional and Sabin IPV and to be strictly serotype-specific. The method is highly sensitive and robust and produces linear results in a wide range of concentrations. We have also found that reference standards used for measuring potency of cIPV and sIPV must be made from respective vaccines. This makes it impossible to cross-calibrate potency reagents made from heterologous vaccine and requires the establishment of a new unit to measure potency of sIPV that is different from conventional D-antigen unit.

Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening.

Hybridoma methods for monoclonal antibody (mAb) cloning are a mainstay of biomedical research, but they are hindered by the need to maintain hybridomas in oligoclonal pools during antibody screening. Here, we describe a system in which hybridomas specifically capture and display the mAbs they secrete: On-Cell mAb Screening (OCMS™). In OCMS™, mAbs displayed on the cell surface can be rapidly assayed for expression level and binding specificity using fluorescent antigens with high-content (image-based) methods or flow cytometry. OCMS™ demonstrated specific mAb binding to poliovirus and rabies virus by forming a cell surface IgG "cap", as a universal assay for anti-viral mAbs. We produced and characterized OCMS™-enabled hybridomas secreting mAbs that neutralize poliovirus and used fluorescence microscopy to identify and clone a human mAb specific for the human N-methyl-D-aspartate receptor. Lastly, we used OCMS™ to assess expression and antigen binding of a recombinant mAb produced in 293T cells. As a novel method to physically associate mAbs with the hybridomas that secrete them, OCMS™ overcomes a central challenge to hybridoma mAb screening and offers new paradigms for mAb discovery and production.

Palliative Care in Lung Cancer: When to Start.

PURPOSE OF REVIEW: Despite recent advances in the care of patients with advanced non-small cell lung cancer (NSCLC), significant morbidity and mortality remains. Symptoms caused by the cancer and its treatments can be profoundly debilitating. Palliative care aims to reduce this burden. In this review, we discuss the definition, purpose, benefits, and optimal timing of palliative care in advanced NSCLC. RECENT FINDINGS: Several studies evaluating the value of early palliative care for patients with advanced NSCLC and other advanced malignancies have identified benefits for patients, caregivers, and health systems. For patients with advanced NSCLC, introduction of palliative care early in the disease course improves quality of life and even overall survival. Early institution of palliative care should become standard of care for patients with advanced NSCLC.

Monoclonal antibodies from a patient with anti-NMDA receptor encephalitis.

OBJECTIVE: Anti-NMDA receptor encephalitis (ANRE) is a potentially lethal encephalitis attributed to autoantibodies against the N-methyl-D-aspartate receptor (NMDAR). We sought to clone and characterize monoclonal antibodies (mAbs) from an ANRE patient. METHODS: We used a hybridoma method to clone two IgG mAbs from a female patient with ANRE without teratoma, and characterized their binding activities on NMDAR-transfected cell lines, cultured primary rat neurons, and mouse hippocampus. We also assessed their effects on voluntary locomotor activity in mice and binding to NMDAR in vivo. RESULTS: The mAbs are structurally distinct and arose from distinct B-cell lineages. They recognize different epitopes on the GluN1 amino terminal domain (ATD), yet both require amino acids important for post-translational modification. Both mAbs bind subsets of GluN1 on cultured rat hippocampal neurons. The 5F5 mAb binds mouse brain hippocampal tissues, and the GluN1 recognized on cultured rat neurons was substantially extra-synaptic. Antibody binding to primary hippocampal neurons induced receptor internalization. The NMDAR inhibitor MK-801 inhibited internalization without preventing mAb binding; AP5 inhibited both mAb binding and internalization. Exposure of mice to the mAbs following permeabilization of the blood brain barrier increased voluntary wheel running activity, similar to low doses of the NMDAR inhibitor, MK-801. INTERPRETATION: These mAbs recapitulate features demonstrated in previous studies of ANRE patient CSF, and exert effects on NMDAR in vitro and in vivo consistent with modulation of NMDAR activity.

Membrane-bound and soluble forms of an NMDA receptor extracellular domain retain epitopes targeted in auto-immune encephalitis.

BACKGROUND: Anti-NMDA receptor encephalitis (ANRE) is a potentially lethal disease attributed to auto-antibodies against the N-methyl-D-aspartate receptor (NMDAR). Full recovery is possible if therapy is initiated early in the disease course. Detection of ANRE antibodies in the cerebrospinal fluid (CSF) is essential for diagnosis. The assays for ANRE-associated IgGs often rely on cells transiently transfected with NMDAR genes. A cell line that stably expresses pathogenic NMDAR epitopes could improve standardization of the assays and provide antigen that could be used in commercial solid state assay systems. RESULTS: We expressed the amino terminal domain (ATD) of the GluN1 NMDAR subunit (NR1) as a fusion protein on the outer plasma membrane of 293T cells, creating a stable cell population (293T-ATD) that is recognized by ANRE patient monoclonal antibodies in flow cytometry and immunofluorescence assays. The ATD fusion protein also contains a Myc tag and a 6XHIS tag, which provide functionality for immunoassays and antigen purification, and a TEV protease site, which allows the ATD domain to be specifically released from the cells in essentially pure form. ATD mobilized from the 293T ATD cell line maintained the pathogenic ANRE epitopes in ELISA binding assays. CSF (3/4) and sera (4/4) from ANRE patients also bound the 293T-ATD cell line, whereas normal CSF and sera did not. CONCLUSIONS: The 293T-ATD cell line is potentially adaptable to a variety of formats to identify antibodies associated with ANRE, including cell-based and soluble antigen formats, and demonstrates a useful method to produce complex proteins for research, drug discovery, and clinical diagnosis.

RBC Adherence of Immune Complexes Containing Botulinum Toxin Improves Neutralization and Macrophage Uptake.

In the paralytic disease botulism, the botulinum neurotoxin (BoNT) passes through the bloodstream to reach and inactivate neuromuscular junctions. Monoclonal antibodies (mAbs) may be useful BoNT countermeasures, as mAb combinations can rapidly clear BoNT from the blood circulation. We have previously shown that the BoNT-neutralizing potency of mAbs can be improved through red blood cell (RBC) immunoadherence. For example, a fusion protein (FP) that adheres biotinylated mAbs to the RBC surface enabled a pair of mAbs to neutralize 5000 LD50 BoNT/A in the mouse protection assay. Here, we added two mAbs to that combination, creating a 4-mAb:FP complex that neutralized 40,000 LD50 BoNT/A in vivo, and analyzed functional correlates of neutralization. The FP enhanced potency of BoNT/A immune complexes, providing the greatest magnitude of benefit to the 4-mAb combination. RBC binding of a BoNT/A complexed with 4-mAb:FP exhibited a bi-phasic clearance process in vivo. Most of the complexes were cleared within five minutes; the rest were cleared gradually over many hours. Peritoneal macrophages showed better uptake of the 4-mAb complex than the 3-mAb complex, and this was not affected by the presence of the FP. However, the addition of RBCs to the 4-mAb:FP BoNT/A doubled macrophage uptake of the complexes. Lastly, the 4-mAb:FP BoNT/A complex synergistically induced M2 macrophage polarization, as indicated by IL-10 expression, whether or not RBCs were present. RBC-targeted immunoadherence through the FP is a potent enhancer of mAb-mediated BoNT/A neutralization in vivo, and can have positive effects on BoNT/A sequestration, immune complex uptake, and macrophage activation.

Characterization of human monoclonal antibodies that neutralize multiple poliovirus serotypes.

Following the eradication of wild poliovirus (PV), achieving and maintaining a polio-free status will require eliminating potentially pathogenic PV strains derived from the oral attenuated vaccine. For this purpose, a combination of non-cross-resistant drugs, such as small molecules and neutralizing monoclonal antibodies (mAbs), may be ideal. We previously isolated chimpanzee and human mAbs capable of neutralizing multiple PV types (cross-neutralization). Here, we describe three additional human mAbs that neutralize types 1 and 2 PV and one mAb that neutralizes all three types. Most bind conformational epitopes and have unusually long heavy chain complementarity determining 3 domains (HC CDR3). We assessed the ability of the mAbs to neutralize A12 escape mutant PV strains, and found that the neutralizing activities of the mAbs were disrupted by different amino acid substitutions. Competitive binding studies further suggested that the specific mAb:PV interactions that enable cross-neutralization differ among mAbs and serotypes. All of the cloned mAbs bind PV in the vicinity of the "canyon", a circular depression around the 5-fold axis of symmetry through which PV recognizes its cellular receptor. We were unable to generate escape mutants to two of the mAbs, suggesting that their epitopes are important for the PV life cycle. These data indicate that PV cross-neutralization involves binding to highly conserved structures within the canyon that binds to the cellular receptor. These may be facilitated by the long HC CDR3 domains, which may adopt alternative binding configurations. We propose that the human and chimpanzee mAbs described here could have potential as anti-PV therapeutics.

A human monoclonal IgG that binds aß assemblies and diverse amyloids exhibits anti-amyloid activities in vitro and in vivo.

Alzheimer's disease (AD) and familial Danish dementia (FDD) are degenerative neurological diseases characterized by amyloid pathology. Normal human sera contain IgG antibodies that specifically bind diverse preamyloid and amyloid proteins and have shown therapeutic potential in vitro and in vivo. We cloned one of these antibodies, 3H3, from memory B cells of a healthy individual using a hybridoma method. 3H3 is an affinity-matured IgG that binds a pan-amyloid epitope, recognizing both Aß and λ Ig light chain (LC) amyloids, which are associated with AD and primary amyloidosis, respectively. The pan-amyloid-binding properties of 3H3 were demonstrated using ELISA, immunohistochemical studies, and competition binding assays. Functional studies showed that 3H3 inhibits both Aß and LC amyloid formation in vitro and abrogates disruption of hippocampal synaptic plasticity by AD-patient-derived soluble Aß in vivo. A 3H3 single-chain variable fragment (scFv) retained the binding specificity of the 3H3 IgG and, when expressed in the brains of transgenic mice using an adeno-associated virus (AAV) vector, decreased parenchymal Aß amyloid deposition in TgCRND8 mice and ADan (Danish Amyloid) cerebral amyloid angiopathy in the mouse model of FDD. These data indicate that naturally occurring human IgGs can recognize a conformational, amyloid-specific epitope and have potent anti-amyloid activities, providing a rationale to test their potential as antibody therapeutics for diverse neurological and other amyloid diseases.

Human monoclonal antibodies that neutralize vaccine and wild-type poliovirus strains.

An essential requirement for eradication of poliomyelitis is the elimination of circulating vaccine derived polioviruses (cVDPV) and polioviruses excreted by chronically infected individuals with immunodeficiencies (iVDPV). As part of a post-eradication risk management strategy, a human monoclonal antibody (mAb) therapeutic could play a role in halting excretion in asymptomatic carriers and could be used, in combination with vaccines and antiviral drugs, to protect polio-exposed individuals. Cross-neutralizing mAbs may be particularly useful, as they would reduce the number of mAbs needed to create a comprehensive PV therapeutic. We cloned a panel of IgG mAbs from OPV-vaccinated, IPV-boosted healthy subjects. Many of the mAbs had potent neutralizing activities against PV wild-type (WT) and Sabin strains, and two of the mAbs, 12F8 and 1E4, were significantly cross-reactive against types 1 and 2 and types 1 and 3, respectively. Mapping the binding epitopes using strains resistant to neutralization (escape mutants) suggested that cross-specific PV binding epitopes may primarily reside within the canyon region, which interacts with the cellular receptor molecule CD155 and the cross-neutralizing chimpanzee/human mAb, A12. Despite their close proximity, the epitopes for the 12F8 and 1E4 mAbs on Sabin 1 were not functionally identical to the A12 epitope. When tested together, 12F8 and 1E4 neutralized a diverse panel of clinically relevant PV strains and did not exhibit interference. Virus mutants resistant to the anti-poliovirus drug V-073 were also neutralized by the mAbs. The 12F8 and 1E4 mAbs may suitable for use as anti-PV therapeutics.