IgE, IgE Receptors and Anti-IgE Biologics: Protein Structures and Mechanisms of Action.

The evolution of IgE in mammals added an extra layer of immune protection at body surfaces to provide a rapid and local response against antigens from the environment. The IgE immune response employs potent expulsive and inflammatory forces against local antigen provocation, at the risk of damaging host tissues and causing allergic disease. Two well-known IgE receptors, the high-affinity FcεRI and low-affinity CD23, mediate the activities of IgE. Unlike other known antibody receptors, CD23 also regulates IgE expression, maintaining IgE homeostasis. This mechanism evolved by adapting the function of the complement receptor CD21. Recent insights into the dynamic character of IgE structure, its resultant capacity for allosteric modulation, and the potential for ligand-induced dissociation have revealed previously unappreciated mechanisms for regulation of IgE and IgE complexes. We describe recent research, highlighting structural studies of the IgE network of proteins to analyze the uniquely versatile activities of IgE and anti-IgE biologics.

Signaling by Antibodies: Recent Progress.

IgG antibodies mediate a diversity of immune functions by coupling of antigen specificity through the Fab domain to signal transduction via Fc-Fc receptor interactions. Indeed, balanced IgG signaling through type I and type II Fc receptors is required for the control of proinflammatory, anti-inflammatory, and immunomodulatory processes. In this review, we discuss the mechanisms that govern IgG-Fc receptor interactions, highlighting the diversity of Fc receptor-mediated effector functions that regulate immunity and inflammation as well as determine susceptibility to infection and autoimmunity and responsiveness to antibody-based therapeutics and vaccines.

Potent efficacy of an IgG-specific endoglycosidase against IgG-mediated pathologies.

Endo-ß-N-acetylglucosaminidases (ENGases) that specifically hydrolyze the Asn297-linked glycan on immunoglobulin G (IgG) antibodies, the major molecular determinant of fragment crystallizable (Fc) γ receptor (FcγR) binding, are exceedingly rare. All previously characterized IgG-specific ENGases are multi-domain proteins secreted as an immune evasion strategy by Streptococcus pyogenes strains. Here, using in silico analysis and mass spectrometry techniques, we identified a family of single-domain ENGases secreted by pathogenic corynebacterial species that exhibit strict specificity for IgG antibodies. By X-ray crystallographic and surface plasmon resonance analyses, we found that the most catalytically efficient IgG-specific ENGase family member recognizes both protein and glycan components of IgG. Employing in vivo models, we demonstrated the remarkable efficacy of this IgG-specific ENGase in mitigating numerous pathologies that rely on FcγR-mediated effector functions, including T and B lymphocyte depletion, autoimmune hemolytic anemia, and antibody-dependent enhancement of dengue disease, revealing its potential for treating and/or preventing a wide range of IgG-mediated diseases in humans.

Antibody effector functions are associated with protection from respiratory syncytial virus.

Respiratory syncytial virus (RSV) infection is a major cause of severe lower respiratory tract infection and death in young infants and the elderly. With no effective prophylactic treatment available, current vaccine candidates aim to elicit neutralizing antibodies. However, binding and neutralization have poorly predicted protection in the past, and accumulating data across epidemiologic cohorts and animal models collectively point to a role for additional antibody Fc-effector functions. To begin to define the humoral correlates of immunity against RSV, here we profiled an adenovirus 26 RSV-preF vaccine-induced humoral immune response in a group of healthy adults that were ultimately challenged with RSV. Protection from infection was linked to opsonophagocytic functions, driven by IgA and differentially glycosylated RSV-specific IgG profiles, marking a functional humoral immune signature of protection against RSV. Furthermore, Fc-modified monoclonal antibodies able to selectively recruit effector functions demonstrated significant antiviral control in a murine model of RSV.

Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope.

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.

Extremely potent human monoclonal antibodies from COVID-19 convalescent patients.

Human monoclonal antibodies are safe, preventive, and therapeutic tools that can be rapidly developed to help restore the massive health and economic disruption caused by the coronavirus disease 2019 (COVID-19) pandemic. By single-cell sorting 4,277 SARS-CoV-2 spike protein-specific memory B cells from 14 COVID-19 survivors, 453 neutralizing antibodies were identified. The most potent neutralizing antibodies recognized the spike protein receptor-binding domain, followed in potency by antibodies that recognize the S1 domain, the spike protein trimer, and the S2 subunit. Only 1.4% of them neutralized the authentic virus with a potency of 1-10 ng/mL. The most potent monoclonal antibody, engineered to reduce the risk of antibody-dependent enhancement and prolong half-life, neutralized the authentic wild-type virus and emerging variants containing D614G, E484K, and N501Y substitutions. Prophylactic and therapeutic efficacy in the hamster model was observed at 0.25 and 4 mg/kg respectively in absence of Fc functions.

Compromised SARS-CoV-2-specific placental antibody transfer.

SARS-CoV-2 infection causes more severe disease in pregnant women compared to age-matched non-pregnant women. Whether maternal infection causes changes in the transfer of immunity to infants remains unclear. Maternal infections have previously been associated with compromised placental antibody transfer, but the mechanism underlying this compromised transfer is not established. Here, we used systems serology to characterize the Fc profile of influenza-, pertussis-, and SARS-CoV-2-specific antibodies transferred across the placenta. Influenza- and pertussis-specific antibodies were actively transferred. However, SARS-CoV-2-specific antibody transfer was significantly reduced compared to influenza- and pertussis-specific antibodies, and cord titers and functional activity were lower than in maternal plasma. This effect was only observed in third-trimester infection. SARS-CoV-2-specific transfer was linked to altered SARS-CoV-2-antibody glycosylation profiles and was partially rescued by infection-induced increases in IgG and increased FCGR3A placental expression. These results point to unexpected compensatory mechanisms to boost immunity in neonates, providing insights for maternal vaccine design.

Compromised Humoral Functional Evolution Tracks with SARS-CoV-2 Mortality.

The urgent need for an effective SARS-CoV-2 vaccine has forced development to progress in the absence of well-defined correlates of immunity. While neutralization has been linked to protection against other pathogens, whether neutralization alone will be sufficient to drive protection against SARS-CoV-2 in the broader population remains unclear. Therefore, to fully define protective humoral immunity, we dissected the early evolution of the humoral response in 193 hospitalized individuals ranging from moderate to severe. Although robust IgM and IgA responses evolved in both survivors and non-survivors with severe disease, non-survivors showed attenuated IgG responses, accompanied by compromised Fcɣ receptor binding and Fc effector activity, pointing to deficient humoral development rather than disease-enhancing humoral immunity. In contrast, individuals with moderate disease exhibited delayed responses that ultimately matured. These data highlight distinct humoral trajectories associated with resolution of SARS-CoV-2 infection and the need for early functional humoral immunity.

Fc Characteristics Mediate Selective Placental Transfer of IgG in HIV-Infected Women.

The placental transfer of maternal IgG is critical for infant protection against infectious pathogens. However, factors that modulate the placental transfer of IgG remain largely undefined. HIV-infected women have impaired placental IgG transfer, presenting a unique "disruption model" to define factors that modulate placental IgG transfer. We measured the placental transfer efficiency of maternal HIV and pathogen-specific IgG in US and Malawian HIV-infected mothers and their HIV-exposed uninfected and infected infants. We examined the role of maternal HIV disease progression, infant factors, placental Fc receptor expression, IgG subclass, and glycan signatures and their association with placental IgG transfer efficiency. Maternal IgG characteristics, such as binding to placentally expressed Fc receptors FcγRIIa and FcγRIIIa, and Fc region glycan profiles were associated with placental IgG transfer efficiency. Our findings suggest that Fc region characteristics modulate the selective placental transfer of IgG, with implications for maternal vaccine design and infant health.

Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.

Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry.

Antibodies and complement are key drivers of thrombosis.

Venous thromboembolism (VTE) is a common, deadly disease with an increasing incidence despite preventive efforts. Clinical observations have associated elevated antibody concentrations or antibody-based therapies with thrombotic events. However, how antibodies contribute to thrombosis is unknown. Here, we show that reduced blood flow enabled immunoglobulin M (IgM) to bind to FcµR and the polymeric immunoglobulin receptor (pIgR), initiating endothelial activation and platelet recruitment. Subsequently, the procoagulant surface of activated platelets accommodated antigen- and FcγR-independent IgG deposition. This leads to classical complement activation, setting in motion a prothrombotic vicious circle. Key elements of this mechanism were present in humans in the setting of venous stasis as well as in the dysregulated immunothrombosis of COVID-19. This antibody-driven thrombosis can be prevented by pharmacologically targeting complement. Hence, our results uncover antibodies as previously unrecognized central regulators of thrombosis. These findings carry relevance for therapeutic application of antibodies and open innovative avenues to target thrombosis without compromising hemostasis.

Breadth of Fc-mediated effector function correlates with clinical immunity following human malaria challenge.

Malaria is a life-threatening disease of global health importance, particularly in sub-Saharan Africa. The growth inhibition assay (GIA) is routinely used to evaluate, prioritize, and quantify the efficacy of malaria blood-stage vaccine candidates but does not reliably predict either naturally acquired or vaccine-induced protection. Controlled human malaria challenge studies in semi-immune volunteers provide an unparalleled opportunity to robustly identify mechanistic correlates of protection. We leveraged this platform to undertake a head-to-head comparison of seven functional antibody assays that are relevant to immunity against the erythrocytic merozoite stage of Plasmodium falciparum. Fc-mediated effector functions were strongly associated with protection from clinical symptoms of malaria and exponential parasite multiplication, while the gold standard GIA was not. The breadth of Fc-mediated effector function discriminated clinical immunity following the challenge. These findings present a shift in the understanding of the mechanisms that underpin immunity to malaria and have important implications for vaccine development.

Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement.

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.

Size-Dependent Segregation Controls Macrophage Phagocytosis of Antibody-Opsonized Targets.

Macrophages protect the body from damage and disease by targeting antibody-opsonized cells for phagocytosis. Though antibodies can be raised against antigens with diverse structures, shapes, and sizes, it is unclear why some are more effective at triggering immune responses than others. Here, we define an antigen height threshold that regulates phagocytosis of both engineered and cancer-specific antigens by macrophages. Using a reconstituted model of antibody-opsonized target cells, we find that phagocytosis is dramatically impaired for antigens that position antibodies >10 nm from the target surface. Decreasing antigen height drives segregation of antibody-bound Fc receptors from the inhibitory phosphatase CD45 in an integrin-independent manner, triggering Fc receptor phosphorylation and promoting phagocytosis. Our work shows that close contact between macrophage and target is a requirement for efficient phagocytosis, suggesting that therapeutic antibodies should target short antigens in order to trigger Fc receptor activation through size-dependent physical segregation.

Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1.

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

mRNA-1273 vaccine-induced antibodies maintain Fc effector functions across SARS-CoV-2 variants of concern.

SARS-CoV-2 mRNA vaccines confer robust protection against COVID-19, but the emergence of variants has generated concerns regarding the protective efficacy of the currently approved vaccines, which lose neutralizing potency against some variants. Emerging data suggest that antibody functions beyond neutralization may contribute to protection from the disease, but little is known about SARS-CoV-2 antibody effector functions. Here, we profiled the binding and functional capacity of convalescent antibodies and Moderna mRNA-1273 COVID-19 vaccine-induced antibodies across SARS-CoV-2 variants of concern (VOCs). Although the neutralizing responses to VOCs decreased in both groups, the Fc-mediated responses were distinct. In convalescent individuals, although antibodies exhibited robust binding to VOCs, they showed compromised interactions with Fc-receptors. Conversely, vaccine-induced antibodies also bound robustly to VOCs but continued to interact with Fc-receptors and mediate antibody effector functions. These data point to a resilience in the mRNA-vaccine-induced humoral immune response that may continue to offer protection from SARS-CoV-2 VOCs independent of neutralization.

A Functional Role for Antibodies in Tuberculosis.

While a third of the world carries the burden of tuberculosis, disease control has been hindered by a lack of tools, including a rapid, point-of-care diagnostic and a protective vaccine. In many infectious diseases, antibodies (Abs) are powerful biomarkers and important immune mediators. However, in Mycobacterium tuberculosis (Mtb) infection, a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach, we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses, such that Ltb infection is associated with unique Ab Fc functional profiles, selective binding to FcγRIII, and distinct Ab glycosylation patterns. Moreover, compared to Abs from Atb, Abs from Ltb drove enhanced phagolysosomal maturation, inflammasome activation, and, most importantly, macrophage killing of intracellular Mtb. Combined, these data point to a potential role for Fc-mediated Ab effector functions, tuned via differential glycosylation, in Mtb control.

A Fc engineering approach to define functional humoral correlates of immunity against Ebola virus.

Protective Ebola virus (EBOV) antibodies have neutralizing activity and induction of antibody constant domain (Fc)-mediated innate immune effector functions. Efforts to enhance Fc effector functionality often focus on maximizing antibody-dependent cellular cytotoxicity, yet distinct combinations of functions could be critical for antibody-mediated protection. As neutralizing antibodies have been cloned from EBOV disease survivors, we sought to identify survivor Fc effector profiles to help guide Fc optimization strategies. Survivors developed a range of functional antibody responses, and we therefore applied a rapid, high-throughput Fc engineering platform to define the most protective profiles. We generated a library of Fc variants with identical antigen-binding fragments (Fabs) from an EBOV neutralizing antibody. Fc variants with antibody-mediated complement deposition and moderate natural killer (NK) cell activity demonstrated complete protective activity in a stringent in vivo mouse model. Our findings highlight the importance of specific effector functions in antibody-mediated protection, and the experimental platform presents a generalizable resource for identifying correlates of immunity to guide therapeutic antibody design.

Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

CD47 Ligation Repositions the Inhibitory Receptor SIRPA to Suppress Integrin Activation and Phagocytosis.

CD47 acts as a "don't eat me" signal that protects cells from phagocytosis by binding and activating its receptor SIPRA on macrophages. CD47 suppresses multiple different pro-engulfment "eat me" signals, including immunoglobulin G (IgG), complement, and calreticulin, on distinct target cells. This complexity has limited understanding of how the "don't eat me" signal is transduced biochemically. Here, we utilized a reconstituted system with a defined set of signals to interrogate the mechanism of SIRPA activation and its downstream targets. CD47 ligation altered SIRPA localization, positioning SIRPA for activation at the phagocytic synapse. At the phagocytic synapse, SIRPA inhibited integrin activation to limit macrophage spreading across the surface of the engulfment target. Chemical reactivation of integrin bypassed CD47-mediated inhibition and rescued engulfment, similar to the effect of a CD47 function-blocking antibody. Thus, the CD47-SIRPA axis suppresses phagocytosis by inhibiting inside-out activation of integrin signaling in the macrophage, with implications to cancer immunotherapy applications.