Antineonatal Fc Receptor Antibody Treatment Ameliorates MOG-IgG–Associated Experimental Autoimmune Encephalomyelitis

Background and ObjectivesMyelin oligodendrocyte glycoprotein antibody–associated disorder (MOGAD) is a rare, autoimmune demyelinating CNS disorder, distinct from multiple sclerosis and neuromyelitis optica spectrum disorder. Characterized by pathogenic immunoglobulin G (IgG) antibodies against MOG, a potential treatment strategy for MOGAD is to reduce circulating IgG levels, e.g., by interference with the IgG recycling pathway mediated by the neonatal Fc receptor (FcRn). Although the optic nerve is often detrimentally involved in MOGAD, the effect of FcRn blockade on the visual pathway has not been assessed. Our objective was to investigate effects of a monoclonal anti-FcRn antibody in murine MOG-IgG–associated experimental autoimmune encephalomyelitis (EAE).MethodsWe induced active MOG35-55 EAE in C57Bl/6 mice followed by the application of a monoclonal MOG-IgG (8-18C5) 10 days postimmunization (dpi). Animals were treated with either a specific monoclonal antibody against FcRn (α-FcRn, 4470) or an isotype-matched control IgG on 7, 10, and 13 dpi. Neurologic disability was scored daily on a 10-point scale. Visual acuity was assessed by optomotor reflex. Histopathologic hallmarks of disease were assessed in the spinal cord, optic nerve, and retina. Immune cell infiltration was visualized by immunohistochemistry, demyelination by Luxol fast blue staining and complement deposition and number of retinal ganglion cells by immunofluorescence.ResultsIn MOG-IgG–augmented MOG35-55 EAE, anti-FcRn treatment significantly attenuated neurologic disability over the course of disease (mean area under the curve and 95% confidence intervals (CIs): α-FcRn [n = 27], 46.02 [37.89–54.15]; isotype IgG [n = 24], 66.75 [59.54–73.96], 3 independent experiments), correlating with reduced amounts of demyelination and macrophage infiltration into the spinal cord. T- and B-cell infiltration and complement deposition remained unchanged. Compared with isotype, anti-FcRn treatment prevented reduction of visual acuity over the course of disease (median cycles/degree and interquartile range: α-FcRn [n = 16], 0.50 [0.48–0.55] to 0.50 [0.48–0.58]; isotype IgG [n = 17], 0.50 [0.49–0.54] to 0.45 [0.39–0.51]).DiscussionWe show preserved optomotor response and ameliorated course of disease after anti-FcRn treatment in an experimental model using a monoclonal MOG-IgG to mimic MOGAD. Selectively targeting FcRn might represent a promising therapeutic approach in MOGAD.

Detection of Antibody Responses Against SARS-CoV-2 in Plasma and Saliva From Vaccinated and Infected Individuals

Antibodies (Abs) are essential for the host immune response against SARS-CoV-2, and all the vaccines developed so far have been designed to induce Abs targeting the SARS-CoV-2 spike. Many studies have examined Ab responses in the blood from vaccinated and infected individuals. However, since SARS-CoV-2 is a respiratory virus, it is also critical to understand the mucosal Ab responses at the sites of initial virus exposure. Here, we examined plasma versus saliva Ab responses in vaccinated and convalescent patients. Although saliva levels were significantly lower, a strong correlation was observed between plasma and saliva total Ig levels against all SARS-CoV-2 antigens tested. Virus-specific IgG1 responses predominated in both saliva and plasma, while a lower prevalence of IgM and IgA1 Abs was observed in saliva. Antiviral activities of plasma Abs were also studied. Neutralization titers against the initial WA1 (D614G), B.1.1.7 (alpha) and B.1.617.2 (delta) strains were similar but lower against the B.1.351 (beta) strain. Spike-specific antibody-dependent cellular phagocytosis (ADCP) activities were also detected and the levels correlated with spike-binding Ig titers. Interestingly, while neutralization and ADCP potencies of vaccinated and convalescent groups were comparable, enhanced complement deposition to spike-specific Abs was noted in vaccinated versus convalescent groups and corresponded with higher levels of IgG1 plus IgG3 among the vaccinated individuals. Altogether, this study demonstrates the detection of Ab responses after vaccination or infection in plasma and saliva that correlate significantly, although Ig isotypic differences were noted. The induced plasma Abs displayed Fab-mediated and Fc-dependent functions with comparable neutralization and ADCP potencies, but a greater capacity to activate complement was elicited upon vaccination.

Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities

Soluble Angiotensin-Converting Enzyme 2 (ACE2) constitutes an attractive antiviral receptor decoy that targets the vulnerable site on SARS-CoV-2 spike (S). Here, using structure-guided approaches, we developed divalent ACE2 molecules by grafting the extracellular ACE2-domain onto a human IgG1 or IgG3 (ACE2-Fc). These ACE2-Fcs harbored structurally validated mutations that enhance S binding and remove enzymatic activity. The lead variant bound tightly to S, mediated in vitro neutralization of SARS-CoV-2 variants of concern (VOCs) with sub-nanomolar IC50 and was capable of robust Fc-effector functions, including antibody-dependent cellular cytotoxicity, phagocytosis and complement deposition. When tested in a stringent K18-hACE2 mouse model, the lead variant prevented or delayed lethal SARS-CoV-2 infection in a prophylactic or therapeutic setting utilizing the combined effect of neutralization and Fc-effector functions. These data confirm the utility of ACE2-Fcs as valuable agents in preventing and eliminating SARS-CoV-2 infection and demonstrate that ACE2-Fc therapeutic activity require Fc-effector functions.

Evidence of Classical Complement Pathway Involvement in a Subset of Patients with Warm Autoimmune Hemolytic Anemia

Introduction: Autoimmune Hemolytic Anemia (AIHA) is caused by autoantibodies that react with red blood cells (RBCs) resulting in predominantly extravascular hemolysis in an FcR and/or complement-dependent manner. In warm AIHA (wAIHA), autoantibodies are generally of the IgG isotype, while in cold agglutinin disease (CAD) they are predominantly of the IgM isotype. It is well established that the classical complement cascade is critical for the pathogenesis of CAD based on therapeutic clinical studies. Published data also suggest that complement activation plays a role in wAIHA, although it is not clear which patients would most benefit from complement-based therapy. To help address this question, we utilized an assay that measures the ability of autoantibodies in patient sera to induce complement deposition on the surface of donor RBCs (based on Meulenbroek, et al., 2015). Methods: Sera were collected retrospectively from 12 wAIHA patients whose direct antiglobulin tests (DAT) were either IgG+/C3+ or IgG+/C3-. Sera retrospectively collected from two CAD patients were used as positive controls. Individual patient sera were examined in the in vitro complement deposition assay using RBCs from type O+ healthy donors. RBCs and sera were incubated at 37 oC in the presence of either EDTA or an inhibitory antibody against C1q as inhibitors of the classical pathway. RBCs were then stained and processed by flow cytometry to determine the level of C4 deposition. Results: Sera from both CAD patients deposited C4 on the surface of ~70% of healthy human RBCs in vitro. Four out of twelve (33%) sera from wAIHA patients displayed this activity, and all four of these patients were identified as IgG+/C3+ on DAT. Complement deposition ranged from ~10-60% of the RBCs in wAIHA, suggesting heterogeneity in antibody activity for complement deposition in sera from wAIHA patients. Addition of EDTA or an inhibitory antibody against C1q fully blocked deposition of C4 on RBCs by wAIHA sera, indicating dependence of the classical complement pathway. These results indicate differences in the frequency of classical pathway involvement in CAD versus wAIHA and may help identify a subset of wAIHA patients most likely to respond to anti-C1q therapy. Conclusions: The hypothesis of classical complement cascade involvement in wAIHA disease in a subset of patients is supported by our results. Critically, complement deposition on the surface of cells by anti-C1q prevented the deposition of a downstream complement marker, C4. Inhibition of C1q has been shown to block activation of all downstream classical complement components, including C3b and C4b involved in extravascular hemolysis and C5b involved in direct cell lysis. The therapeutic potential of blocking classical complement pathway activity in wAIHA is currently being evaluated in an ongoing Phase 2 interventional trial (NCT04691570) assessing efficacy of an anti-C1q drug candidate in wAIHA patients, focusing on those with evidence of classical complement pathway activity. Disclosures Teigler: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Low: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Rose: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Cahir-Mcfarland: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Yednock: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Kroon: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Keswani: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Barcellini: Novartis: Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria; Incyte: Membership on an entity's Board of Directors or advisory committees.

A morphological transformation in respiratory syncytial virus leads to enhanced complement deposition

The complement system is a critical host defense against infection, playing a protective role that can also enhance disease if dysregulated. Although many consequences of complement activation during viral infection are well established, mechanisms that determine the extent to which viruses activate complement remain elusive. Here, we investigate complement activation by human respiratory syncytial virus (RSV), a filamentous respiratory pathogen that causes significant morbidity and mortality. By engineering a strain of RSV harboring tags on the surface glycoproteins F and G, we are able to monitor opsonization of single RSV particles using fluorescence microscopy. These experiments reveal an antigenic hierarchy, where antibodies that bind toward the apex of F in either the pre- or postfusion conformation activate the classical pathway whereas other antibodies do not. Additionally, we identify an important role for virus morphology in complement activation: as viral filaments age, they undergo a morphological transformation which lowers the threshold for complement deposition through changes in surface curvature. Collectively, these results identify antigenic and biophysical characteristics of virus particles that contribute to the formation of viral immune complexes, and suggest models for how these factors may shape disease severity and adaptive immune responses to RSV.

RNA sequencing reveals induction of specific renal inflammatory pathways in a rat model of malignant hypertension

In malignant hypertension, far more severe kidney injury occurs than in the “benign” form of the disease. The role of high blood pressure and the renin–angiotensin–aldosterone system is well recognized, but the pathogenesis of the renal injury of malignant hypertension (MH) remains incompletely understood. Using the rat model of two-kidney, one-clip renovascular hypertension in which some but not all animals develop MH, we performed a transcriptomic analysis of gene expression by RNA sequencing to identify transcriptional changes in the kidney cortex specific for MH. Differential gene expression was assessed in three groups: MH, non-malignant hypertension (NMH), and normotensive, sham-operated controls. To distinguish MH from NMH, we considered two factors: weight loss and typical renovascular lesions. Mean blood pressure measured intraarterially was elevated in MH (220 ± 6.5 mmHg) as well as in NMH (192 ± 6.4 mmHg), compared to controls (119 ± 1.7 mmHg, p < 0.05). Eight hundred eighty-six genes were exclusively regulated in MH only. Principal component analysis revealed a separated clustering of the three groups. The data pointed to an upregulation of many inflammatory mechanisms in MH including pathways which previously attracted relatively little attention in the setting of hypertensive kidney injury: Transcripts from all three complement activation pathways were upregulated in MH compared to NMH but not in NMH compared with controls; immunohistochemistry confirmed complement deposition in MH exclusively. The expression of chemokines attracting neutrophil granulocytes (CXCL6) and infiltration of myeloperoxidase-positive cells were increased only in MH rats. The data suggest that these pathways, especially complement deposition, may contribute to kidney injury under MH. Key messages The most severe hypertension-induced kidney injury occurs in malignant hypertension. In a rat model of malignant hypertension, we assessed transcriptional responses in the kidney exposed to high blood pressure. A broad stimulation of inflammatory mechanisms was observed, but a few specific pathways were activated only in the malignant form of the disease, notably activation of the complement cascades. Complement inhibitors may alleviate the thrombotic microangiopathy of malignant hypertension even in the absence of primary complement abnormalities.

Antibody Attributes that Predict the Neutralization and Effector Function of Polyclonal Responses to SARS-CoV-2

While antibodies provide significant protection from SARS-CoV-2 infection and disease sequelae, the specific attributes of the humoral response that contribute to immunity are incompletely defined. In this study, we employ machine learning to relate characteristics of the polyclonal antibody response raised by natural infection to diverse antibody effector functions and neutralization potency with the goal of generating both accurate predictions of each activity based on antibody response profiles as well as insights into antibody mechanisms of action. To this end, antibody-mediated phagocytosis, cytotoxicity, complement deposition, and neutralization were accurately predicted from biophysical antibody profiles in both discovery and validation cohorts. These predictive models identified SARS-CoV-2-specific IgM as a key predictor of neutralization activity whose mechanistic relevance was supported experimentally by depletion. Validated models of how different aspects of the humoral response relate to antiviral antibody activities suggest desirable attributes to recapitulate by vaccination or other antibody-based interventions.

Case Report: In Situ Expression of a Proliferation-Inducing Ligand in Neuromyelitis Optica

A proliferation inducing ligand (APRIL) mediates a key role in the generation and survival of antibody-inducing plasmocytes. Based on this, APRIL has been targeted in autoimmune diseases including multiple sclerosis (MS) and optic neuritis (ON). In MS lesions, APRIL has a new cellular target, the reactive astrocyte and mediates an immunosuppressive activity. Here, we analyzed APRIL expression in a case of neuromyelitis optica (NMO), another autoimmune neurodegenerative disease, showing selective aquaporin-4 depletion in the spinal cord, complement deposition and infiltration of polymorphonuclear cells. We analyzed by immunohistochemistry the presence of APRIL-producing cells, plasmocytes, astrocytes and the localization of secreted APRIL in a lesion from NMO. Plasmocytes were present close to APRIL-producing cells in meninges. However, our main observation was that APRIL targets reactive astrocytes in this lesion of NMO similarly to MS.

Entamoeba histolytica develops resistance to complement deposition and lysis after acquisition of human complement regulatory proteins through trogocytosis

Entamoeba histolytica is the cause of amoebiasis. The trophozoite (amoeba) form of this parasite is capable of invading the intestine, and can disseminate through the bloodstream to other organs. The mechanisms that allow amoebae to evade complement deposition during dissemination have not been well characterized. We previously discovered a novel complement-evasion mechanism employed by E. histolytica. E. histolytica ingests small bites of living human cells in a process termed trogocytosis. We demonstrated that amoebae were protected from lysis by human serum following trogocytosis of human cells, and that amoebae acquired and displayed human membrane proteins from the cells they ingested. Here, we aimed to define how amoebae are protected from complement lysis after performing trogocytosis. We found that amoebae were protected from complement lysis after ingestion of both human Jurkat T cells and red blood cells, and that the level of protection correlated with the amount of material ingested. Trogocytosis of human cells led to a reduction in deposition of C3b on the surface of amoebae. We asked whether display of human complement regulators is involved in amoebic protection, and found that CD59 was displayed by amoebae after trogocytosis. Deletion of a single complement regulatory protein, CD59 or CD46, from Jurkat cells was not sufficient to alter amoebic protection. Removal of all GPI-anchored proteins, including CD59 and CD55, from the surface of amoebae that had undergone trogocytosis suggested that multiple, redundant complement regulators mediate amoebic protection. These studies shed light on a novel strategy for immune evasion by a pathogen.