Antibodies produced in response to a foreign antigen are characterized by polyclonality, not only in the diverse epitopes to which their variable domains bind but also in the various effector molecules to which their constant regions (Fc domains) engage. Thus, the antibody's Fc domain mediates diverse effector activities by engaging two distinct classes of Fc receptors (type I and type II) on the basis of the two dominant conformational states that the Fc domain may adopt. These conformational states are regulated by the differences among antibody subclasses in their amino acid sequence and by the complex, biantennary Fc-associated N-linked glycan. Here we discuss the diverse downstream proinflammatory, anti-inflammatory and immunomodulatory consequences of the engagement of type I and type II Fc receptors in the context of infectious, autoimmune, and neoplastic disorders.

After the emergence of pandemic influenza viruses in 1957, 1968, and 2009, existing seasonal viruses were observed to be replaced in the human population by the novel pandemic strains. We have previously hypothesized that the replacement of seasonal strains was mediated, in part, by a population-scale boost in antibodies specific for conserved regions of the hemagglutinin stalk and the viral neuraminidase. Numerous recent studies have shown the role of stalk-specific antibodies in neutralization of influenza viruses; the finding that stalk antibodies can effectively neutralize virus alters the existing dogma that influenza virus neutralization is mediated solely by antibodies that react with the globular head of the viral hemagglutinin. The present study explores the possibility that stalkspecific antibodies were boosted by infection with the 2009 H1N1 pandemic virus and that those antibodies could have contributed to the disappearance of existing seasonal H1N1 influenza virus strains. To study stalk-specific antibodies, we have developed chimeric hemagglutinin constructs that enable the measurement of antibodies that bind the hemagglutinin protein and neutralize virus but do not have hemagglutination inhibition activity. Using these chimeric hemagglutinin reagents, we show that infection with the 2009 pandemic H1N1 virus elicited a boost in titer of virus-neutralizing antibodies directed against the hemagglutinin stalk. In addition, we describe assays that can be used to measure influenza virus-neutralizing antibodies that are not detected in the traditional hemagglutination inhibition assay.cross-reactivity | cross-protection | subtype

The influenza virus hemagglutinin molecule possesses a globular head domain that mediates receptor binding and a stalk domain at the membrane-proximal region. We generated functional influenza viruses expressing chimeric hemagglutinins encompassing a variety of globular head and stalk combinations, not only from different hemagglutinin subtypes but also from different hemagglutinin phylogenetic groups. These chimeric recombinant viruses possess growth properties similar to those of wildtype influenza viruses and can be used as reagents to measure domain-specific antibodies in virological and immunological assays.

SUMMARY Protective vaccines elicit high affinity, neutralizing antibodies by selection of somatically hypermutated B-cell antigen receptors (BCR) on immune complexes (ICs). This implicates Fc-Fc receptor (FcR) interactions in affinity maturation, which, in turn, are determined by IgG subclass and Fc glycan composition within ICs. Trivalent influenza virus vaccination elicited regulation of anti-hemagglutinin (HA) IgG subclass and Fc glycans, with abundance of sialylated Fc glycans (sFc) predicting quality of vaccine response. We show that sFcs drive BCR affinity selection by binding the Type-II FcR CD23, thus upregulating the inhibitory FcγRIIB on activated B-cells. This elevates the threshold requirement for BCR signaling, resulting in B-cell selection for higher affinity BCR. Immunization with sFc HA ICs elicited protective, high affinity IgGs against the conserved stalk of the HA. These results reveal a novel, endogenous pathway for affinity maturation that can be exploited for eliciting high affinity, broadly neutralizing antibodies through immunization with sialylated immune complexes.

Immunoglobulins recognize and clear microbial pathogens and toxins through the coupling of variable region specificity to Fc-triggered cellular activation. These proinflammatory activities are regulated, thus avoiding the pathogenic sequelae of uncontrolled inflammation by modulating the composition of the Fc-linked glycan. Upon sialylation, the affinities for Fcγ receptors are reduced, whereas those for alternative cellular receptors, such as dendritic cellspecific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)/CD23, are increased. We demonstrate that sialylation induces significant structural alterations in the Cγ2 domain and propose a model that explains the observed changes in ligand specificity and biological activity. By analogy to related complexes formed by IgE and its evolutionarily related Fc receptors, we conclude that this mechanism is general for the modulation of antibody-triggered immune responses, characterized by a shift between an "open" activating conformation and a "closed" antiinflammatory state of antibody Fc fragments. This common mechanism has been targeted by pathogens to avoid host defense and offers targets for therapeutic intervention in allergic and autoimmune disorders.conformational change | sialylated IgG Fc I gG and IgE mediate their proinflammatory properties through the crosslinking of the 1:1 complex of the Fc receptor (FcR) monomer in the Fc dimer cleft (1, 2). By contrast, both IgG and IgE can engage a second class of receptors, the evolutionarily related, C-type lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) (3) and CD23 (4), respectively, resulting in anti-inflammatory and immunosuppressive responses (5, 6). The structural basis for the ability of IgE to interact either with one or the other of these two disparate classes of receptors has recently been defined (7). The intrinsic flexibility of the IgE Ce3 domain results in both open and closed conformations of the IgE Fc, resulting in the binding of either FceRI or CD23, respectively. Binding of either receptor induces an allosteric change in the IgE Fc to the alternative conformation, thus precluding the interaction with the other receptor (7). Binding of IgE to the type II, C-type lectin CD23 is neither carbohydrate-nor calcium-dependent, mediated exclusively through protein-protein interactions, generating a 2:1 complex of CD23 with the Ce3-Ce4 interface (7). DC-SIGN is a structurally homologous, calcium-dependent, carbohydratebinding, type II lectin, tightly linked to CD23 on chromosome 19 (8), displaying ligand specificity for mannose-containing glycoconjugates and fucose-containing Lewis antigens. Binding of DC-SIGN to IgG requires that the complex, biantennary glycan, attached to the evolutionarily conserved glycosylation site Asn-297 and enclosed within the cavity formed by the Cγ2 domains of the A and B chains of the Fc dimer, be processed to the α2,6 sialylated form (9, 10). Importantly, no evidence has been found for DC-SIGN binding to sialylate...

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 pro-inflammatory, 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 vaccine responses.

Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype have recently emerged from avian zoonotic reservoirs to cause fatal human disease. Adaptation of HPAI virus RNA-dependent RNA polymerase (PB1, PB2, and PA proteins) and nucleoprotein (NP) to interactions with mammalian host proteins is thought to contribute to the efficiency of viral RNA synthesis and to disease severity. While proteomics experiments have identified a number of human proteins that associate with H1N1 polymerases and/or viral ribonucleoprotein (vRNP), how these host interactions might regulate influenza virus polymerase functions and host adaptation has been largely unexplored. We took a functional genomics (RNA interference [RNAi]) approach to assess the roles of a network of human proteins interacting with influenza virus polymerase proteins in viral polymerase activity from prototype H1N1 and H5N1 viruses. A majority (18 of 31) of the cellular proteins tested, including RNA-binding (DDX17, DDX5, NPM1, and hnRNPM), stress (PARP1, DDB1, and Ku70/86), and intracellular transport proteins, were required for efficient activity of both H1N1 and H5N1 polymerases. NXP2 and NF90 antagonized both polymerases, and six more RNA-associated proteins exhibited strain-specific phenotypes. Remarkably, 12 proteins differentially regulated H5N1 polymerase according to PB2 genotype at mammalian-adaptive residue 627. Among these, DEAD box RNA helicase DDX17/p72 facilitated efficient human-adapted (627K) H5N1 virus mRNA and viral RNA (vRNA) synthesis in human cells. Likewise, the chicken DDX17 homologue was required for efficient avian (627E) H5N1 infection in chicken DF-1 fibroblasts, suggesting that this conserved virus-host interaction contributes to PB2-dependent host species specificity of influenza virus and ultimately to the outcome of human HPAI infections.

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