The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising "universal" influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.T he discovery and ongoing characterization of broadly neutralizing antibodies (bnAbs) that bind to the hemagglutinin (HA) stalk domain of influenza A viruses (IAVs) has galvanized promising new efforts to generate a universal influenza virus vaccine (1). A number of studies have now firmly established that stalk-specific bnAbs, normally present in low quantities, can be boosted substantially in humans following exposure to HA subtypes with antigenically foreign head domains (2-8). Sequential vaccination of animals with chimeric HAs or with "headless" vaccine constructs have effectively recapitulated the boosting of bnAbs observed in humans after exposure to foreign HAs, and also are protective against heterologous and heterosubtypic IAV challenge (9-15). These strategies are now being tested as promising "universal" influenza virus vaccine candidates (16).Whereas traditional strain-specific antibodies neutralize virus by inhibiting receptor binding, stalk-binding bnAbs neutralize virus by distinct postbinding mechanisms (17). A direct comparison of in vitro neutralization has revealed that strain-specific mAbs that inhibit receptor binding tend to be more potent at neutralizing virus compared with monoclonal stalk-binding bnAbs (18, 19); however, this difference is minimized in the context of a polyclonal response (18). In addition to virus neutralization,...

The aim of candidate universal influenza vaccines is to provide broad protection against influenza A and B viruses. Studies have demonstrated that broadly reactive antibodies require Fc–Fc gamma receptor interactions for optimal protection; however, the innate effector cells responsible for mediating this protection remain largely unknown. Here, we examine the roles of alveolar macrophages, natural killer cells, and neutrophils in antibody-mediated protection. We demonstrate that alveolar macrophages play a dominant role in conferring protection provided by both broadly neutralizing and non-neutralizing antibodies in mice. Our data also reveal the potential mechanisms by which alveolar macrophages mediate protection in vivo, namely antibody-induced inflammation and antibody-dependent cellular phagocytosis. This study highlights the importance of innate effector cells in establishing a broad-spectrum antiviral state, as well as providing a better understanding of how multiple arms of the immune system cooperate to achieve an optimal antiviral response following influenza virus infection or immunization.

Spider silks show unique combinations of strength, toughness, extensibility, and energy absorption. To date, it has been difficult to obtain spider silk-like mechanical properties using non-protein approaches. Here, we report on an artificial spider silk produced by the water-evaporation-induced self-assembly of hydrogel fibre made from polyacrylic acid and silica nanoparticles. The artificial spider silk consists of hierarchical core-sheath structured hydrogel fibres, which are reinforced by ion doping and twist insertion. The fibre exhibits a tensile strength of 895 MPa and a stretchability of 44.3%, achieving mechanical properties comparable to spider silk. The material also presents a high toughness of 370 MJ m−3 and a damping capacity of 95%. The hydrogel fibre shows only ~1/9 of the impact force of cotton yarn with negligible rebound when used for impact reduction applications. This work opens an avenue towards the fabrication of artificial spider silk with applications in kinetic energy buffering and shock-absorbing.

Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

Influenza virus strain-specific monoclonal antibodies (mAbs) provide protection independent of Fc gamma receptor (FcγR) engagement. In contrast, optimal in vivo protection achieved by broadly reactive mAbs requires Fc-FcγR engagement. Most strain-specific mAbs target the head domain of the viral hemagglutinin (HA), whereas broadly reactive mAbs typically recognize epitopes within the HA stalk. This observation has led to questions regarding the mechanism regulating the activation of Fc-dependent effector functions by broadly reactive antibodies. To dissect the molecular mechanism responsible for this dichotomy, we inserted the FLAG epitope into discrete locations on HAs. By characterizing the interactions of several FLAG-tagged HAs with a FLAG-specific antibody, we show that in addition to Fc-FcγR engagement mediated by the FLAG-specific antibody, a second intermolecular bridge between the receptor-binding region of the HA and sialic acid on effector cells is required for optimal activation. Inhibition of this second molecular bridge, through the use of an F(ab′) 2 or the mutation of the sialic acid-binding site, renders the Fc-FcγR interaction unable to optimally activate effector cells. Our findings indicate that broadly reactive mAbs require two molecular contacts to possibly stabilize the immunologic synapse and potently induce antibody-dependent cell-mediated antiviral responses: (i) the interaction between the Fc of a mAb bound to HA with the FcγR of the effector cell and (ii) the interaction between the HA and its sialic acid receptor on the effector cell. This concept might be broadly applicable for protective antibody responses to viral pathogens that have suitable receptors on effector cells. hemagglutinin | influenza virus | antibody-dependent cell-mediated immunity | broadly reactive antibodies | Fcγ receptor