Revealing the Specificity of Human H1 Influenza A Viruses to Complex N-Glycans

Influenza virus infection remains a threat to human health since viral hemagglutinins are constantly drifting, escaping infection and vaccine-induced antibody responses. Viral hemag-glutinins from different viruses display variability in glycan recognition. In this context, recent H3N2 viruses have specificity for alpha 2,6 sialylated branched N-glycans with at least three N- acetyllactosamine units (tri-LacNAc). In this work, we combined glycan arrays and tissue binding analyses with nuclear magnetic resonance experiments to characterize the glycan specificity of a family of H1 variants, including the one responsible for the 2009 pandemic outbreak. We also analyzed one engineered H6N1 mutant to understand if the preference for tri-LacNAc motifs could be a general trend in human-type receptor-adapted viruses. In addition, we developed a new NMR approach to perform competition experiments between glycans with similar compositions and different lengths. Our results point out that pandemic H1 viruses differ from previous seasonal H1 viruses by a strict preference for a minimum of di-LacNAc structural motifs.R.P.d.V. is a recipient of an ERC Starting grant from the European Commission (802780) and a Beijerinck Premium of the Royal Dutch Academy of Sciences. The glycan array setup was supported by the Netherlands Organization for Scientific Research (NWO, TOP-PUNT 718.015.003 to G.-J.P.H.B.). Dr. Lin Liu (CCRC) and Dr. Margreet A . Wolfert (Utrecht University) developed, printed, and validated the glycan microarray. We would like to thank Nikoloz Nemanichvili for technical assistance. A.C. acknowledges funding from Agencia Estatal de Investigacion "Spanish Ministry of Science and Innovation" (MICINN) project PID2019-105237GB-I00. J.P.C. acknowledges funding by the Spanish MICINN, grant no. RTI2018-095588-B-I00 (co-funded by the European Regional Development Fund/European Social Fund, "Invest-ing in your future"). JJB also tha n k s funding by the European Research Council (RECGLYCANMR, Advanced grant no. 788143), the Agencia Estatal de Investigacion (Spain) for grants RTI2018-094751-B-C21 and C22 and PDI2021-1237810B-C21 and C22, and CIBERES, an initiative of the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. The NMR spectra were acquired at the NMR service of CIBMargarita Salas and in the NMR faci l i t y of the UCM. We also acknowledge Prof. Robert Woods group for sending us the coordinates of a glycan-hemagglut i n i n model

Structures of the Inhibitory Receptor Siglec-8 in Complex with a High-Affinity Sialoside Analogue and a Therapeutic Antibody

Human sialic acid binding immunoglobulin-like lectin-8 (Siglec-8) is an inhibitory receptor that triggers eosinophil apoptosis and can inhibit mast cell degranulation when engaged by specific monoclonal antibodies (mAbs) or sialylated ligands. Thus, Siglec-8 has emerged as a critical negative regulator of inflammatory responses in diverse diseases, such as allergic airway inflammation. Herein, we have deciphered the molecular recognition features of the interaction of Siglec-8 with the mAb lirentelimab (2C4, under clinical development) and with a sialoside mimetic with the potential to suppress mast cell degranulation. The three-dimensional structure of Siglec-8 and the fragment antigen binding (Fab) portion of the anti-Siglec-8 mAb 2C4, solved by X-ray crystallography, reveal that 2C4 binds close to the carbohydrate recognition domain (V-type Ig domain) on Siglec-8. We have also deduced the binding mode of a high-affinity analogue of its sialic acid ligand (9-N-napthylsufonimide-Neu5Ac, NSANeuAc) using a combination of NMR spectroscopy and X-ray crystallography. Our results show that the sialoside ring of NSANeuAc binds to the canonical sialyl binding pocket of the Siglec receptor family and that the high affinity arises from the accommodation of the NSA aromatic group in a nearby hydrophobic patch formed by the N-terminal tail and the unique G–G′ loop. The results reveal the basis for the observed high affinity of this ligand and provide clues for the rational design of the next generation of Siglec-8 inhibitors. Additionally, the specific interactions between Siglec-8 and the N-linked glycans present on the high-affinity receptor FcεRIα have also been explored by NMR.This work was supported by operating grant PID2019-107770RA-I00 (J.E.-O.) from the Agencia Estatal Investigación of Spain and by the European Research Council (ERC-2017-AdG, 788143-RECGLYCANMR to J.J.-B.). We also thank the Marie-Skłodowska-Curie actions (ITN Glytunes grant agreement no. 956758 to J.E.-O and ITN BactiVax under grant agreement no. 860325 to U.A.). Additional funding was provided by CIBER, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain. We also thank the Ikerbasque Basque Foundation of Science and the Spanish Ministry of Economy, Industry and Competitiveness (for the postdoctoral contract Juan de la Cierva Incorporación to J.E-O). X-ray diffraction experiments described in this paper were performed using the XALOC synchrotron beamline at ALBA (Spain) and PXIII in Swiss Light Source (Switzerland)

Revealing the Specificity of Human H1 Influenza A Viruses to Complex N-Glycans

Influenza virus infection remains a threat to human health since viral hemagglutinins are constantly drifting, escaping infection and vaccine-induced antibody responses. Viral hemagglutinins from different viruses display variability in glycan recognition. In this context, recent H3N2 viruses have specificity for α2,6 sialylated branched N-glycans with at least three N-acetyllactosamine units (tri-LacNAc). In this work, we combined glycan arrays and tissue binding analyses with nuclear magnetic resonance experiments to characterize the glycan specificity of a family of H1 variants, including the one responsible for the 2009 pandemic outbreak. We also analyzed one engineered H6N1 mutant to understand if the preference for tri-LacNAc motifs could be a general trend in human-type receptor-adapted viruses. In addition, we developed a new NMR approach to perform competition experiments between glycans with similar compositions and different lengths. Our results point out that pandemic H1 viruses differ from previous seasonal H1 viruses by a strict preference for a minimum of di-LacNAc structural motifs

A Siglec-like sialic-acid-binding motif revealed in an adenovirus capsid protein

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of transmembrane receptors that are well documented to play roles in regulation of innate and adaptive immune responses. To see whether the features that define the molecular recognition of sialic acid were found in other sialic-acid-binding proteins, we analyzed 127 structures with bound sialic acids found in the Protein Data Bank database. Of these, the canine adenovirus 2-fiber knob protein showed close local structural relationship to Siglecs despite low sequence similarity. The fiber knob harbors a noncanonical sialic-acid recognition site, which was then explored for detailed specificity using a custom glycan microarray comprising 58 diverse sialosides. It was found that the adenoviral protein preferentially recognizes the epitope Neu5Acalpha2-3[6S]Galbeta1-4GlcNAc, a structure previously identified as the preferred ligand for Siglec-8 in humans and Siglec-F in mice. Comparison of the Siglec and fiber knob sialic-acid-binding sites reveal conserved structural elements that are not clearly identifiable from the primary amino acid sequence, suggesting a Siglec-like sialic-acid-binding motif that comprises the consensus features of these proteins in complex with sialic acid

Major antigenic site B of human influenza H3N2 viruses has an evolving local fitness landscape

Antigenic drift of influenza virus hemagglutinin (HA) is enabled by facile evolvability. However, HA antigenic site B, which has become immunodominant in recent human H3N2 influenza viruses, is also evolutionarily constrained by its involvement in receptor binding. Here, we employ deep mutational scanning to probe the local fitness landscape of HA antigenic site B in six different human H3N2 strains spanning from 1968 to 2016. We observe that the fitness landscape of HA antigenic site B can be very different between strains. Sequence variants that exhibit high fitness in one strain can be deleterious in another, indicating that the evolutionary constraints of antigenic site B have changed over time. Structural analysis suggests that the local fitness landscape of antigenic site B can be reshaped by natural mutations via modulation of the receptor-binding mode. Overall, these findings elucidate how influenza virus continues to explore new antigenic space despite strong functional constraints

A Siglec-like sialic-acid-binding motif revealed in an adenovirus capsid protein

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of transmembrane receptors that are well documented to play roles in regulation of innate and adaptive immune responses. To see whether the features that define the molecular recognition of sialic acid were found in other sialic-acid-binding proteins, we analyzed 127 structures with bound sialic acids found in the Protein Data Bank database. Of these, the canine adenovirus 2-fiber knob protein showed close local structural relationship to Siglecs despite low sequence similarity. The fiber knob harbors a noncanonical sialic-acid recognition site, which was then explored for detailed specificity using a custom glycan microarray comprising 58 diverse sialosides. It was found that the adenoviral protein preferentially recognizes the epitope Neu5Acalpha2-3[6S]Galbeta1-4GlcNAc, a structure previously identified as the preferred ligand for Siglec-8 in humans and Siglec-F in mice. Comparison of the Siglec and fiber knob sialic-acid-binding sites reveal conserved structural elements that are not clearly identifiable from the primary amino acid sequence, suggesting a Siglec-like sialic-acid-binding motif that comprises the consensus features of these proteins in complex with sialic acid

High affinity sialoside ligands of myelin associated glycoprotein

Myelin associated glycoprotein (Siglec-4) is a myelin adhesion receptor, that is, well established for its role as an inhibitor of axonal outgrowth in nerve injury, mediated by binding to sialic acid containing ligands on the axonal membrane. Because disruption of myelin–ligand interactions promotes axon outgrowth, we have sought to develop potent ligand based inhibitors using natural ligands as scaffolds. Although natural ligands of MAG are glycolipids terminating in the sequence NeuAcα2–3Galβ1–3(±NeuAcα2−6)GalNAcβ-R, we previously established that synthetic O-linked glycoprotein glycans with the same sequence α-linked to Thr exhibited ∼1000-fold increased affinity (∼1 μM). Attempts to increase potency by introducing a benzoylamide substituent at C-9 of the α2–3 sialic acid afforded only a two-fold increase, instead of increases of >100-fold observed for other sialoside ligands of MAG. Surprisingly, however, introduction of a 9-N-fluoro-benzoyl substituent on the α2–6 sialic acid increased affinity 80-fold, resulting in a potent inhibitor with a Kd of 15 nM. Docking this ligand to a model of MAG based on known crystal structures of other siglecs suggests that the Thr positions the glycan such that aryl substitution of the α2–3 sialic acid produces a steric clash with the GalNAc, while attaching an aryl substituent to the other sialic acid positions the substituent near a hydrophobic pocket that accounts to the increase in affinity

Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis

Antibodies confer humoral immunity but can also be harmful when they target an autoantigen, alloantigen, allergen, or biotherapeutic. New strategies are needed for antigen-specific suppression of undesired antibody responses, particularly to T cell–dependent protein antigens, because they elicit T cell help. Here we show that liposomal nanoparticles, displaying both antigen and glycan ligands of the inhibitory coreceptor CD22, induce a tolerogenic program that selectively causes apoptosis in mouse and human B cells. These SIGLEC-engaging tolerance-inducing antigenic liposomes (STALs, where SIGLEC is defined as sialic acid–binding Ig-like lectin) induced robust antigen-specific tolerance to protein antigens in mice, preventing subsequent immune response to challenge with the same antigen. Since development of inhibitory antibodies to FVIII is a serious problem in treatment of hemophilia A patients, we investigated the potential of this approach for inducing tolerance to FVIII in a hemophilia mouse model. STALs prevented formation of inhibitory FVIII antibodies, allowing for effective administration of FVIII to hemophilia mice to prevent bleeding. These findings suggest that STALs could be used to eliminate or prevent harmful B cell–mediated immune responses