Salmonella Typhi assembles and secretes two forms of typhoid toxin by using two receptor-binding subunits, PltB and PltC. Unlike PltB typhoid toxin, little is known about the tropism and functional consequences of PltC typhoid toxin. Here, we report that PltC typhoid toxin has hepatobiliary tropism through the binding of PltC subunit to sulfated glycans on liver sinusoidal endothelial cells and gallbladder epithelial cells. Critical bacterial and mammalian cell factors involved are PltC R109 residue and carbohydrate sulfotransferases CHST2/4. One notable effect associated with the hepatobiliary tropism of PltC typhoid toxin is a reduction in bile acids, consequently promoting S. Typhi pathogenicity in infected mice. Similarly, bile acids serve as anti-S. Typhi infectivity agents at the cellular level, as bile acids inhibit invasion of mammalian cells. These findings highlight a distinct mechanism used by a bacterial exotoxin promoting the pathogenicity of the cognate bacteria and offer insights into the development of antivirulence agents against PltC typhoid toxin and/or S. Typhi.

Human H3N2 influenza viruses, introduced during the 1968 pandemic, have evolved to recognize human-type sialic acid-containing receptors (Neu5Acα2-6Gal) extended with at least three LacNAc (Galβ1-4GlcNAc) repeats. To investigate this restriction in the context of virus attachment to the airway epithelium, we comprehensively analyzed the glycome of human nasal and tracheal epithelial cells. Using a synthetic N-glycan library that reflects the structural diversity of the human airway glycome, we found that only bi-antennary N-glycans with extended human-type receptors on at least one branch serve as receptors for the recent H3 hemagglutinins (HAs). Such receptors are found on tracheal epithelium but are deficient in nasal epithelium. Immunofluorescence analysis on human trachea reveals that recent H3 HAs preferentially attach to ciliated cells, consistent with single-cell RNA sequencing analysis indicating that these cells express glycosyltransferases that produce extended glycan chains. These findings suggest that H3N2 viruses have developed a tropism for tracheal ciliated cells. (146 words) ### Competing Interest Statement The authors have declared no competing interest. National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, AI 114730, Contract No. 75N93021C00015 The Wellcome Trust, Grant 082098

The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode. The receptor binding mode of recent human H3N2 hemagglutinin has evolved due to mutations G186D and D190N, which epistatically interact and co-emerged in clades 3C.2a1b.1a and 3C.2a1b.2a2.

Hemagglutinins (HAs) from human influenza viruses descend from avian progenitors that bind α2-3-linked sialosides and must adapt to glycans with α2-6-linked sialic acids on human airway cells to transmit within the human population. Since their introduction during the 1968 pandemic, H3N2 viruses have evolved over the past five decades to preferentially recognize human α2-6-sialoside receptors that are elongated through addition of poly-LacNAc. We show that more recent H3N2 viruses now make increasingly complex interactions with elongated receptors while continuously selecting for strains maintaining this phenotype. This change in receptor engagement is accompanied by an extension of the traditional receptor-binding site to include residues in key antigenic sites on the surface of HA trimers. These results help explain the propensity for selection of antigenic variants, leading to vaccine mismatching, when H3N2 viruses are propagated in chicken eggs or cells that do not contain such receptors.

Influenza A viruses in swine have considerable genetic diversity and continue to pose a pandemic threat to humans due to a potential lack of population level immunity. Here we describe a pipeline to characterize and triage influenza viruses for their pandemic risk and examine the pandemic potential of two widespread swine origin viruses. Our analysis reveals that a panel of human sera collected from healthy adults in 2020 has no cross-reactive neutralizing antibodies against a α-H1 clade strain (α-swH1N2) but do against a γ-H1 clade strain. The α-swH1N2 virus replicates efficiently in human airway cultures and exhibits phenotypic signatures similar to the human H1N1 pandemic strain from 2009 (H1N1pdm09). Furthermore, α-swH1N2 is capable of efficient airborne transmission to both naïve ferrets and ferrets with prior seasonal influenza immunity. Ferrets with H1N1pdm09 pre-existing immunity show reduced α-swH1N2 viral shedding and less severe disease signs. Despite this, H1N1pdm09-immune ferrets that became infected via the air can still onward transmit α-swH1N2 with an efficiency of 50%. These results indicate that this α-swH1N2 strain has a higher pandemic potential, but a moderate level of impact since there is reduced replication fitness and pathology in animals with prior immunity.

Sulfated N-glycans are present in many glycoproteins, which are implicated in playing important roles in biological recognition processes. Here, we report the systematic chemoenzymatic synthesis of a library of sulfated and sialylated biantennary N-glycans and assess their binding to Siglecs and glycan-specific antibodies that recognize them as glycan ligands. The combined use of three human sulfotransferases, GlcNAc-6-O-sulfotransferase (CHST2), Gal-3-O-sulfotransferase (Gal3ST1), and keratan sulfate Gal-6-O-sulfotransferase (CHST1), resulted in asymmetric and symmetric branch-selective sulfation of the GlcNAc and/or Gal moieties of N-glycans. The extension of the sugar chain using alpha-2,3- and alpha-2,6-sialyltransferases afforded the sulfated and sialylated N-glycans. These synthetic glycans with different patterns of sulfation and sialylation were evaluated for binding to selected Siglecs and sulfoglycan-specific antibodies using glycan microarrays. The results confirm previously documented glycan-recognizing properties and further reveal novel specificities for these glycan-binding proteins, demonstrating the utility of the library for assessing the specificity of glycan-binding proteins recognizing sulfated and sialylated glycans.

Clade 2.3.4.4b highly pathogenic H5N1 avian influenza (HPAI) viruses started circulating widely in lactating dairy cattle in the United States at the end of 2023. Avian influenza viruses enter cells after binding to glycan receptors with terminally linked α2-3 sialic acid, whereas human influenza viruses typically bind to glycan receptors terminally linked α2-6 sialic acid in the upper respiratory tract. Here, we evaluated the receptor binding properties of hemagglutinin (HA) trimers from a clade 2.3.4.4b avian isolate (A/American Wigeon/South Carolina/22-000345-001/2021) and a cattle isolate (A/dairy cattle/Texas/24-008749-002-v/2024). Using two different methods, we found that both of the 2.3.4.4b H5s bound efficiently to glycan receptors with terminally linked α2-3 sialic acid with no detectable binding to glycan receptors with terminally linked α2-6 sialic acid. Our data suggest that clade 2.3.4.4b H5N1 viruses bind poorly to human receptors. It will be important to continue evaluating receptor binding properties of these viruses as they evolve in cattle.

In 2024, several human infections with highly pathogenic clade 2.3.4.4b bovine influenza H5N1 viruses in the United States raised concerns about their capability for bovine-to-human or even human-to-human transmission. In this study, analysis of the hemagglutinin (HA) from the first-reported human-infecting bovine H5N1 virus (A/Texas/37/2024, Texas) revealed avian-type receptor binding preference. Notably, a Gln 226 Leu substitution switched Texas HA binding specificity to human-type receptors, which was enhanced when combined with an Asn 224 Lys mutation. Crystal structures of the Texas HA with avian receptor analog LSTa and its Gln 226 Leu mutant with human receptor analog LSTc elucidated the structural basis for this preferential receptor recognition. These findings highlight the need for continuous surveillance of emerging mutations in avian and bovine clade 2.3.4.4b H5N1 viruses.

Vaccination strategies aimed at maturing broadly neutralizing antibodies (bnAbs) from naïve precursors are hindered by unusual features that characterize these Abs, including insertions and deletions (indels). Longitudinal studies of natural HIV infection cases shed light on the complex processes underlying bnAb development and have suggested a role for superinfection as a potential enhancer of neutralization breadth. Here we describe the development of a potent bnAb lineage that was elicited by two founder viruses to inform vaccine design. The V3-glycan targeting bnAb lineage (PC39-1) was isolated from subtype C-infected IAVI Protocol C elite neutralizer, donor PC39, and is defined by the presence of multiple independent insertions in CDRH1 that range from 1-11 amino acids in length. Memory B cell members of this lineage are predominantly atypical in phenotype yet also span the class-switched and antibody-secreting cell compartments. Development of neutralization breadth occurred concomitantly with extensive recombination between founder viruses before each virus separated into two distinct population "arms" that evolved independently to escape the PC39-1 lineage. Ab crystal structures show an extended CDRH1 that can help stabilize the CDRH3. Overall, these findings suggest that early exposure of the humoral system to multiple related Env molecules could promote the induction of bnAbs by focusing Ab responses to conserved epitopes.

Ganglioside sugars, as Tumour-Associated Carbohydrate Antigens (TACAs), are long-proposed targets for vaccination and therapeutic antibody production, but their self-like character imparts immunorecessive characteristics that classical vaccination approaches have to date failed to overcome. One prominent TACA, the glycan component of ganglioside GM3 (GM3g), is over-expressed on diverse tumours. To probe the limits of glycan tolerance, we used protein editing methods to display GM3g in systematically varied non-native presentation modes by attachment to carrier protein lysine sidechains using diverse chemical linkers. We report here that such presentation creates glycoconjugates that are strongly immunogenic in mice and elicit robust antigen-specific IgG responses specific to GM3g. Characterisation of this response by antigen-specific B cell cloning and phylogenetic and functional analyses suggests that such display enables the engagement of a highly restricted naïve B cell class with a defined germline configuration dominated by members of the IGHV2 subgroup. Strikingly, structural analysis reveals that glycan features appear to be recognised primarily by antibody CDRH1/2, and despite the presence of an antigen-specific Th response and B cell somatic hypermutation, we found no evidence of affinity maturation towards the antigen. Together these findings suggest a ‘reach-through’ model in which glycans, when displayed in non-self formats of sufficient distance from a conjugate backbone, may engage ‘glycan ready’ V-region motifs encoded in the germline. Structural constraints define why, despite engaging the trisaccharide, antibodies do not bind natively-presented glycans, such as when linked to lipid GM3. Our findings provide an explanation for the long-standing difficulties in raising antibodies reactive with native TACAs, and provide a possible template for rational vaccine design against this and other TACA antigens. Highlights GM3g synthetically coupled via a longer, orthogonal (from backbone) glycoconjugate (LOG) presentation format (thioethyl-lysyl-amidine) display elicits high-titre IgG responses in mice. The germinal centre experience of LOG glycoconjugate-specific B cell responses is directly influenced by the protein backbone. Structural characterisation of the antibody response to LOGs reveals highly restricted germline-encoded glycan-engaging motifs that mediate GM3g recognition. Failure of antibodies to bind the native trisaccharide highlights barriers to be overcome for the rational design of anti-TACA antibodies.

Influenza viruses circulated at very low levels during the beginning of the COVID-19 pandemic, and population immunity against these viruses is low. An H3N2 strain (3C.2a1b.2a2) with a hemagglutinin (HA) that has several substitutions relative to the 2021-22 H3N2 vaccine strain is dominating the 2021-22 Northern Hemisphere influenza season. Here, we show that one of these substitutions eliminates a key glycosylation site on HA and alters sialic acid binding. Using glycan array profiling, we show that the 3C.2a1b.2a2 H3 maintains binding to an extended biantennary sialoside and replicates to high titers in human airway cells. We find that antibodies elicited by the 2021-22 Northern Hemisphere influenza vaccine poorly neutralize the 3C.2a1b.2a2 H3N2 strain. Together, these data indicate that 3C.2a1b.2a2 H3N2 viruses efficiently replicate in human cells and escape vaccine-elicited antibodies.

Siglecs are a family of sialic acid-binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Ac alpha 2-3(6-O-sulfo)Gal beta 1-4GlcNAc (6'-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer's disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid-binding proteins.

Influenza viruses have circulated at very low levels during the COVID-19 pandemic, and population immunity against these viruses is low. Influenza virus cases have been increasing in the Northern Hemisphere involving an H3N2 strain (3C.2a1b.2a2) with a hemagglutinin (HA) that has several substitutions relative to the 2021-2022 H3N2 vaccine strain. Here, we show that one of these substitutions eliminates a key glycosylation site on HA and alters sialic acid binding. Using glycan array profiling, we show that the 3C.2a1b.2a2 H3 maintains binding to an extended bi-antennary sialoside and replicates to high titers in human airway cells. We found that antibodies elicited by the 2021-2022 Northern Hemisphere influenza vaccine poorly neutralize the new H3N2 strain. Together, these data indicate that 3C.2a1b.2a2 H3N2 viruses efficiently replicate in human cells and could potentially cause an antigenic mismatch if they continue to circulate at high levels during the 2021-2022 influenza season.

Due to the very low concentration and polyclonal nature of IgE in peripheral blood, the molecular detail underlying allergy to red meat has eluded study. Human monoclonal antibodies (mAbs) from subjects with red meat allergy will now allow comprehensive characterization of both the antibodies and allergens involved in this response. We employ a human B cell hybridoma method to immortalize IgE encoding B cells from peripheral blood mononuclear cells (PBMCs) of subjects with red meat allergy, generating naturally-occurring human IgE mAbs. B cell cultures were screened in an unbiased manner for IgE production without regard to specificity. Isolated IgE mAbs were tested for binding to alpha-gal using ImmunoCAP, bovine thyroglobulin, and cetuximab, and their epitopes mapped using a 600 glycan microarray developed by the Consortium for Functional Glycomics. We isolated two IgE mAbs specific to the alpha-gal allergen from a subject with red meat allergy. The frequency of alpha-gal specific IgE-encoding B cells was exceedingly low at approximately 4.0 cells/108 PBMCs. The two antibody sequences exhibit a significant degree of somatic hypermutation, bind to alpha-gal allergens, and exhibit different fine specificity for glycan epitopes involving both classical alpha-gal (Galα1-3Gal) and non-alpha-gal moieties. We have generated the first human hybridomas secreting naturally-occurring alpha-gal specific IgE mAbs from a subject with red meat allergy. The goal of our work is to improve upon our molecular understanding of the human IgE antibody response, which will provide insights needed for the design of better immunotherapies and allergy vaccines.

H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human-type receptors, increase hemagglutinin (HA) stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability, and receptor specificity. None of the tested substitutions resulted in a clear step toward a human-adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally.

In 2014, an outbreak of avian A/H10N7 influenza virus occurred among seals along North-European coastal waters, significantly impacting seal populations. Here, we examine the cross-species transmission and mammalian adaptation of this influenza A virus, revealing changes in the hemagglutinin surface protein that increase stability and receptor binding. The seal A/H10N7 virus was aerosol or respiratory droplet transmissible between ferrets. Compared with avian H10 hemagglutinin, seal H10 hemagglutinin showed stronger binding to the human-type sialic acid receptor, with preferential binding to α2,6-linked sialic acids on long extended branches. In X-ray structures, changes in the 220-loop of the receptor-binding pocket caused similar interactions with human receptor as seen for pandemic strains. Two substitutions made seal H10 hemagglutinin more stable than avian H10 hemagglutinin and similar to human hemagglutinin. Consequently, identification of avian-origin influenza viruses across mammals appears critical to detect influenza A viruses posing a major threat to humans and other mammals.

Avian influenza viruses (AIVs) recognize sialic acid linked α2,3 to galactose (SAα2,3Gal) glycans as receptors. In this study, the interactions between hemagglutinins (HAs) of AIVs and sulfated SAα2,3Gal glycans were analyzed to clarify the molecular basis of interspecies transmission of AIVs from ducks to chickens. It was revealed that E190V and N192D substitutions of the HA increased the recovery of viruses derived from an H6 duck virus isolate, A/duck/Hong Kong/960/1980 (H6N2), in chickens. Recombinant HAs from an H6 chicken virus, A/chicken/Tainan/V156/1999 (H6N1), bound to sulfated SAα2,3Gal glycans, whereas the HAs from an H6 duck virus did not. Binding preference of mutant HAs revealed that an E190V substitution is critical for the recognition of sulfated SAα2,3Gal glycans. These results suggest that the binding of the HA from H6 AIVs to sulfated SAα2,3Gal glycans explains a part of mechanisms of interspecies transmission of AIVs from ducks to chickens.

Hemagglutinins (HAs) from human influenza viruses adapt to bind α2-6-linked sialosides, overcoming a receptor-defined species barrier distinct from the α2-3 specificity of avian virus progenitors. Additionally, human-adapted HAs gain glycosylation sites over time, although their biological function is poorly defined. Using quantitative glycomic analysis, we show that HAs from human pandemic viruses exhibit significant proportions of high-mannose type N-linked glycans throughout the head domain. By contrast, poorly adapted avian-origin HAs contain predominately complex-type glycans, which have greater structural diversity. Although oligomannose levels vary, they are present in all tested recombinant HAs and whole viruses and can be specifically targeted for universal detection. The positions of high-mannose glycosites on the HA of human H1N1 and H3N2 strains are conserved. Additionally, high-mannose-binding lectins possess a broad capacity to neutralize and prevent infection with contemporary H3N2 strains. These findings reveal the biological significance of HA glycosylation and therapeutic potential of targeting these structures.

The Siglec family of cell surface receptors have emerged as attractive targets for cell-directed therapies due to their restricted expression on immune cells, endocytic properties, and ability to modulate receptor signaling. Human Siglec-8, for instance, has been identified as a therapeutic target for the treatment of eosinophil and mast cell disorders. A promising strategy to target Siglecs involves the use of liposomal nanoparticles with a multivalent display of Siglec ligands. A key challenge for this approach is the identification of a high affinity ligand for the target Siglec. Here, we report the development of a ligand of Siglec-8 and its closest murine functional orthologue Siglec-F that is capable of targeting liposomes to cells expressing Siglec-8 or -F. A glycan microarray library of synthetic 9-N-sulfonyl sialoside analogues was screened to identify potential lead compounds. The best ligand, 9-N-(2-naphthyl-sulfonyl)-Neu5Acα2-3-[6-O-sulfo]-Galβ1-4GlcNAc (6'-O-sulfo NSANeu5Ac) combined the lead 2-naphthyl sulfonyl C-9 substituent with the preferred sulfated scaffold. The ligand 6'-O-sulfo NSANeu5Ac was conjugated to lipids for display on liposomes to evaluate targeted delivery to cells. Targeted liposomes showed strong in vitro binding/uptake and selectivity to cells expressing Siglec-8 or -F and, when administered to mice, exhibit in vivo targeting to Siglec-F+ eosinophils.