Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA-receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA-sfGFP proteins was studied using glycan arrays and tissue staining. The HA-sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA-sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.

Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells-primarily eosinophils and mast cells-where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose-acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

Viruses employ elaborate strategies to coopt the cellular processes they require to replicate while simultaneously thwarting host antiviral responses. In many instances, how this is accomplished remains poorly understood. Here, we identify a protein, F17 encoded by cytoplasmically replicating poxviruses, that binds and sequesters Raptor and Rictor, regulators of mammalian target of rapamycin complexes mTORC1 and mTORC2, respectively. This disrupts mTORC1-mTORC2 crosstalk that coordinates host responses to poxvirus infection. During infection with poxvirus lacking F17, cGAS accumulates together with endoplasmic reticulum vesicles around the Golgi, where activated STING puncta form, leading to interferon-stimulated gene expression. By contrast, poxvirus expressing F17 dysregulates mTOR, which localizes to the Golgi and blocks these antiviral responses in part through mTOR-dependent cGAS degradation. Ancestral conservation of Raptor/Rictor across eukaryotes, along with expression of F17 across poxviruses, suggests that mTOR dysregulation forms a conserved poxvirus strategy to counter cytosolic sensing while maintaining the metabolic benefits of mTOR activity.

Wild birds harbor a large gene pool of influenza A viruses that have the potential to cause influenza pandemics. Foreseeing and understanding this potential is important for effective surveillance. Our phylogenetic and geographic analyses revealed the global prevalence of avian influenza virus genes whose proteins differ only a few amino acids from the 1918 pandemic influenza virus, suggesting that 1918-like pandemic viruses may emerge in the future. To assess this risk, we generated and characterized a virus composed of avian influenza viral segments with high homology to the 1918 virus. This virus exhibited pathogenicity in mice and ferrets higher than that in an authentic avian influenza virus. Further, acquisition of seven amino acid substitutions in the viral polymerases and the hemagglutinin surface glycoprotein conferred respiratory droplet transmission to the 1918-like avian virus in ferrets, demonstrating that contemporary avian influenza viruses with 1918 virus-like proteins may have pandemic potential.

Low pathogenic H7N9 influenza viruses have recently evolved to become highly pathogenic, raising concerns of a pandemic, particularly if these viruses acquire efficient human-to-human transmissibility. We compared a low pathogenic H7N9 virus with a highly pathogenic isolate, and two of its variants that represent neuraminidase inhibitor-sensitive and -resistant subpopulations detected within the isolate. The highly pathogenic H7N9 viruses replicated efficiently in mice, ferrets, and/or nonhuman primates, and were more pathogenic in mice and ferrets than the low pathogenic H7N9 virus, with the exception of the neuraminidase inhibitor-resistant virus, which showed mild-to-moderate attenuation. All viruses transmitted among ferrets via respiratory droplets, and the neuraminidase-sensitive variant killed several of the infected and exposed animals. Neuraminidase inhibitors showed limited effectiveness against these viruses in vivo, but the viruses were susceptible to a polymerase inhibitor. These results suggest that the highly pathogenic H7N9 virus has pandemic potential and should be closely monitored.

The effectiveness of the annual influenza vaccine has declined in recent years, especially for the H3N2 component, and is a concern for global public health. A major cause for this lack in effectiveness has been attributed to the egg-based vaccine production process. Substitutions on the hemagglutinin glycoprotein (HA) often arise during virus passaging that change its antigenicity and hence vaccine effectiveness. Here, we characterize the effect of a prevalent substitution, L194P, in egg-passaged H3N2 viruses. X-ray structural analysis reveals that this substitution surprisingly increases the mobility of the 190-helix and neighboring regions in antigenic site B, which forms one side of the receptor binding site (RBS) and is immunodominant in recent human H3N2 viruses. Importantly, the L194P substitution decreases binding and neutralization by an RBS-targeted broadly neutralizing antibody by three orders of magnitude and significantly changes the HA antigenicity as measured by binding of human serum antibodies. The receptor binding mode and specificity are also altered to adapt to avian receptors during egg passaging. Overall, these findings help explain the low effectiveness of the seasonal vaccine against H3N2 viruses, and suggest that alternative approaches should be accelerated for producing influenza vaccines as well as isolating clinical isolates.

H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries and have caused repeated human infections in Asia since 1998. To evaluate the potential threat of H9N2 viruses to humans, we investigated the replication and transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT) H9N2 viruses, isolated from different avian species from 1988 through 2003, were tested in vivo and found to replicate in ferrets. However these viruses achieved mild peak viral titers in nasal washes when compared to those observed with a human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact ferrets, however no aerosol transmission was detected in the virus displaying the most efficient direct contact transmission. A leucine (Leu) residue at amino acid position 226 in the hemagglutinin (HA) receptor-binding site (RBS), responsible for human virus-like receptor specificity, was found to be important for the transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human reassortant virus, which contains the surface glycoprotein genes from an H9N2 virus and the six internal genes of a human H3N2 virus, showed enhanced replication and efficient transmission to direct contacts. Although no aerosol transmission was observed, the virus replicated in multiple respiratory tissues and induced clinical signs similar to those observed with the parental human H3N2 virus. Our results suggest that the establishment and prevalence of H9N2 viruses in poultry pose a significant threat for humans.

One goal of regenerative medicine is to repair damaged tissue. This requires not only generating new cells of the proper phenotype, but also selecting for those that properly integrate into sites of injury. In our laboratory we are using a cell-migration-based in vivo selection system to generate antibodies that induce cells to both differentiate and selectively localize to different tissues. Here we describe an antibody that induces bone marrow stem cells to differentiate into microglia-like cells that traffic to the brain where they organize into typical networks. Interestingly, in the APP/PS1 Alzheimer's disease mouse model, these induced microglia-like cells are found at sites of plaque formation and significantly reduce their number. These results raise the intriguing question as to whether one can use such antibody-induced differentiation of stem cells to essentially recapitulate embryogenesis in adults to discover cells that can regenerate damaged organ systems.

The hemagglutinin (HA) receptor-binding site (RBS) in human influenza A viruses is critical for attachment to host cells, which imposes a functional constraint on its natural evolution. On the other hand, being part of the major antigenic sites, the HA RBS of human H3N2 viruses needs to constantly mutate to evade the immune system. From large-scale mutagenesis experiments, we here show that several of the natural RBS substitutions become integrated into an extensive epistatic network that prevents substitution reversion. X-ray structural analysis reveals the mechanistic consequences as well as changes in the mode of receptor binding. Further studies are necessary to elucidate whether such entrenchment limits future options for immune escape or adversely affect long-term viral fitness.

CD33 is an immunomodulatory receptor linked to Alzheimer's disease (AD) susceptibility via regulation of phagocytosis in microglia. Divergent features between human CD33 (hCD33) and murine CD33 (mCD33) include a unique transmembrane lysine in mCD33 and cytoplasmic tyrosine in hCD33. The functional consequences of these differences in restraining phagocytosis remains poorly understood. Using a new αmCD33 monoclonal antibody, we show that mCD33 is expressed at high levels on neutrophils and low levels on microglia. Notably, cell surface expression of mCD33 is entirely dependent on Dap12 due to an interaction with the transmembrane lysine in mCD33. In RAW264.7 cultured macrophages, BV-2 cultured microglia, primary neonatal and adult microglia, uptake of cargo - including aggregated Aβ1-42 - is not altered upon genetic ablation of mCD33. Alternatively, deletion of hCD33 in monocytic cell lines increased cargo uptake. Moreover, transgenic mice expressing hCD33 in the microglial cell lineage showed repressed cargo uptake in primary microglia. Therefore, mCD33 and hCD33 have divergent roles in regulating phagocytosis, highlighting the importance of studying hCD33 in AD susceptibility.

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.

Sialoadhesin (Sn, Siglec-1, CD169) is a member of the sialic acid binding Ig-like lectin (siglec) family expressed on macrophages. Its macrophage specific expression makes it an attractive target for delivering antigens to tissue macrophages via Sn-mediated endocytosis. Here we describe a novel approach for delivering antigens to macrophages using liposomal nanoparticles displaying high affinity glycan ligands of Sn. The Sn-targeted liposomes selectively bind to and are internalized by Sn-expressing cells, and accumulate intracellularly over time. Our results show that ligand decorated liposomes are specific for Sn, since they are taken up by bone marrow derived macrophages that are derived from wild type but not Sn(-/-) mice. Importantly, the Sn-targeted liposomes dramatically enhance the delivery of antigens to macrophages for presentation to and proliferation of antigen-specific T cells. Together, these data provide insights into the potential of cell-specific targeting and delivery of antigens to intracellular organelles of macrophages using Sn-ligand decorated liposomal nanoparticles.

The high pathogenicity of H5N1 viruses in sporadic infections of humans has raised concerns for its potential to acquire the ability to transmit between humans and emerge as a highly pathogenic pandemic virus. Because avian and human influenza viruses differ in their specificity for recognition of their host cell receptors, receptor specificity represents one barrier for efficient transmission of avian viruses in human hosts. Over the last century, each influenza virus pandemic has coincided with the emergence of virus with an immunologically distinct hemagglutinin exhibiting a 'human-type' receptor specificity, distinct from that of viruses with the same hemagglutinin circulating in zoonotic species. Recent studies suggest that it is possible for H5N1 to acquire human type receptor specificity, but this has not occurred in nature. This review covers what is known about the molecular basis for the switch between avian and human-type receptor specificity for influenza viruses that have successfully adapted to man, the potential for H5N1 to evolve to human-type receptor specificity and its relevance to pandemic risk.

Antiangiogenesis therapies are now part of the standard repertoire of cancer therapies, but the mechanisms for the proliferation and survival of endothelial cells are not fully understood. Although endothelial cells are covered with a glycocalyx, little is known about how endothelial glycosylation regulates endothelial functions. Here, we show that alpha2,6-sialic acid is necessary for the cell-surface residency of platelet endothelial cell adhesion molecule (PECAM), a member of the immunoglobulin superfamily that plays multiple roles in cell adhesion, mechanical stress sensing, antiapoptosis, and angiogenesis. As a possible underlying mechanism, we found that the homophilic interactions of PECAM in endothelial cells were dependent on alpha2,6-sialic acid. We also found that the absence of alpha2,6-sialic acid down-regulated the tyrosine phosphorylation of PECAM and recruitment of Src homology 2 domain-containing protein-tyrosine phosphatase 2 and rendered the cells more prone to mitochondrion-dependent apoptosis, as evaluated using PECAM- deficient endothelial cells. The present findings open up a new possibility that modulation of glycosylation could be one of the promising strategies for regulating angiogenesis.

The glycans displayed on mammalian cells can differ markedly from those on microbes. Such differences could, in principle, be 'read' by carbohydrate-binding proteins, or lectins. We used glycan microarrays to show that human intelectin-1 (hIntL-1) does not bind known human glycan epitopes but does interact with multiple glycan epitopes found exclusively on microbes: β-linked D-galactofuranose (β-Galf), D-phosphoglycerol-modified glycans, heptoses, D-glycero-D-talo-oct-2-ulosonic acid (KO) and 3-deoxy-D-manno-oct-2-ulosonic acid (KDO). The 1.6-Å-resolution crystal structure of hIntL-1 complexed with β-Galf revealed that hIntL-1 uses a bound calcium ion to coordinate terminal exocyclic 1,2-diols. N-acetylneuraminic acid (Neu5Ac), a sialic acid widespread in human glycans, has an exocyclic 1,2-diol but does not bind hIntL-1, probably owing to unfavorable steric and electronic effects. hIntL-1 marks only Streptococcus pneumoniae serotypes that display surface glycans with terminal 1,2-diol groups. This ligand selectivity suggests that hIntL-1 functions in microbial surveillance.

New technologies are urgently required for rapid surveillance of the current H5N1 avian influenza A outbreaks to gauge the potential for adaptation of the virus to the human population, a crucial step in the emergence of pandemic influenza virus strains. Owing to the species-specific nature of the interaction between the virus and host glycans, attention has recently focused on novel glycan array technologies that can rapidly assess virus receptor specificity and the potential emergence of human-adapted H5N1 viruses.

Broadly neutralizing antibodies (bnAbs) to HIV delineate vaccine targets and are prophylactic and therapeutic agents. Some of the most potent bnAbs target a quaternary epitope at the apex of the surface HIV envelope (Env) trimer. Using cryo-electron microscopy, we solved the atomic structure of an apex bnAb, PGT145, in complex with Env. We showed that the long anionic HCDR3 of PGT145 penetrated between glycans at the trimer 3-fold axis, to contact peptide residues from all three Env protomers, and thus explains its highly trimer-specific nature. Somatic hypermutation in the other CDRs of PGT145 were crucially involved in stabilizing the structure of the HCDR3, similar to bovine antibodies, to aid in recognition of a cluster of conserved basic residues hypothesized to facilitate trimer disassembly during viral entry. Overall, the findings exemplify the creative solutions that the human immune system can evolve to recognize a conserved motif buried under a canopy of glycans.

In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAcα2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAcα2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding.

Intestinal microfold (M) cells are epithelial cells primarily present on Peyer's patches (PPs) in the small intestine. The ability of M cells to shuttle antigens into the PP for appropriate immune responses makes M cells a target for next-generation oral vaccine delivery. In this regard, discovery of M cell-specific receptors are of great interest, which could act as molecular tags for targeted delivery of cargo to M cells. Here, using a monoclonal antibody we generated to the Sialic acid-binding immunoglobulin-like lectin F (Siglec-F), we show that Siglec-F is expressed on mouse M cells in the small intestine. Immunohistochemical analysis of the PP tissue sections shows that Siglec-F is expressed on the surface of the M cell membrane exposed to the intestinal lumen. Anti-Siglec-F antibody injected into the mouse small intestine bound to M cells, demonstrating the potential to target M cells via Siglec-F.

As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.