Glyco-engineered MDCK cells display preferred receptors of H3N2 influenza absent in eggs used for vaccines.

Evolution of human H3N2 influenza viruses driven by immune selection has narrowed the receptor specificity of the hemagglutinin (HA) to a restricted subset of human-type (Neu5Acα2-6 Gal) glycan receptors that have extended poly-LacNAc (Galß1-4GlcNAc) repeats. This altered specificity has presented challenges for hemagglutination assays, growth in laboratory hosts, and vaccine production in eggs. To assess the impact of extended glycan receptors on virus binding, infection, and growth, we have engineered N-glycan extended (NExt) cell lines by overexpressing ß3-Ν-acetylglucosaminyltransferase 2 in MDCK, SIAT, and hCK cell lines. Of these, SIAT-NExt cells exhibit markedly increased binding of H3 HAs and susceptibility to infection by recent H3N2 virus strains, but without impacting final virus titers. Glycome analysis of these cell lines and allantoic and amniotic egg membranes provide insights into the importance of extended glycan receptors for growth of recent H3N2 viruses and relevance to their production for cell- and egg-based vaccines.

Infection of swine ex vivo tissues with avian viruses including H7N9 and correlation with glycomic analysis.

OBJECTIVES: Swine have been regarded as intermediate hosts in the spread of influenza from birds to humans but studies of the sialylated glycans that comprise their respiratory tract have not been extensively studied in the past. This study analyzed the sialylated N-glycan and O-glycan profile of swine trachea and lung and correlated this with ex-vivo infection of swine explants with avian influenza viruses. SAMPLE: Lungs and tracheal samples were obtained from normal farm and laboratory raised swine and used for ex vivo infection as well as mass spectrometric analysis. Infection of the ex vivo tissues used high pathogenic and low pathogenic avian viruses including the novel H7N9 virus that emerged in China in early 2013. MAIN OUTCOME MEASURES: Assessment of successful replication was determined by TCID50 as well as virus immunohistochemistry. The N-glycan and O-glycan profiles were measured by MALDI-TOF and sialylated linkages were determined by sialidase treatment. Lectin binding histochemistry was also performed on formalin fixed tissue samples with positive binding detected by chromogen staining. RESULTS: The swine respiratory tract glycans differed from the human respiratory tact glycans in two main areas. There was a greater abundance of Gal-α-Gal linkages resulting in a relative decrease in sialylated glycans. The swine respiratory tract also had a greater proportion of glycans containing Neu5Gc and Siaα2-6 glycans than the human respiratory tract. Infection with avian viruses was confined primarily to lung bronchioles rather than trachea and parenchyma. CONCLUSIONS: In contrast to previous studies we found that there was not as much expression of Siaα2-3 glycans on the surface of the trachea. Infection of Siaα2-3 binding avian viruses was restricted to the lower respiratory tract bronchioles. This finding may diminish the ability of the swine to act as an intermediary in the transmission of avian viruses to humans.

Glycomic analysis of human respiratory tract tissues and correlation with influenza virus infection.

The first step in influenza infection of the human respiratory tract is binding of the virus to sialic (Sia) acid terminated receptors. The binding of different strains of virus for the receptor is determined by the α linkage of the sialic acid to galactose and the adjacent glycan structure. In this study the N- and O-glycan composition of the human lung, bronchus and nasopharynx was characterized by mass spectrometry. Analysis showed that there was a wide spectrum of both Sia α2-3 and α2-6 glycans in the lung and bronchus. This glycan structural data was then utilized in combination with binding data from 4 of the published glycan arrays to assess whether these current glycan arrays were able to predict replication of human, avian and swine viruses in human ex vivo respiratory tract tissues. The most comprehensive array from the Consortium for Functional Glycomics contained the greatest diversity of sialylated glycans, but was not predictive of productive replication in the bronchus and lung. Our findings indicate that more comprehensive but focused arrays need to be developed to investigate influenza virus binding in an assessment of newly emerging influenza viruses.

An expression system for screening of proteins for glycan and protein interactions.

Here we describe a versatile high-throughput expression system that permits genome-wide screening of type 1 membrane and secreted proteins for interactions with glycans and proteins using both cell-expressed and soluble forms of the expressed proteins. Based on Gateway cloning methodology, we have engineered a destination vector that directs expression of enhanced green fluorescent protein (EGFP)-tagged proteins at the cell surface via a glycosylphosphatidylinositol tail. The EGFP fusion proteins can then be cleaved with PreScission protease to release soluble forms of proteins that can be optionally biotinylated. We demonstrate the utility of this cloning and expression system for selected low-affinity membrane lectins from the siglec family of sialic acid-binding immunoglobulin-like lectins, for the glycosaminoglycan-binding proteins FGF-1 and BACE, and for the heterotypic adhesion molecules JAM-B and JAM-C. Cell-expressed proteins can be evaluated for glycan interactions using polyvalent soluble glycan probes and for protein interactions using either cells or soluble proteins. Following cleavage from the cell surface, proteins were complexed in solution and sufficient avidity was achieved to measure weak protein-glycan and weak protein-protein interactions using glycan arrays and surface plasmon resonance, respectively.

Glycan analysis and influenza A virus infection of primary swine respiratory epithelial cells: the importance of NeuAc{alpha}2-6 glycans.

To better understand influenza virus infection of pigs, we examined primary swine respiratory epithelial cells (SRECs, the primary target cells of influenza viruses in vivo), as a model system. Glycomic profiling of SRECs by mass spectrometry revealed a diverse range of glycans terminating in sialic acid or GalαGal. In terms of sialylation, α2-6 linkage was more abundant than α2-3, and NeuAc was more abundant than NeuGc. Virus binding and infection experiments were conducted to determine functionally important glycans for influenza virus infection, with a focus on recently emerged swine viruses. Infection of SRECs with swine and human viruses resulted in different infectivity levels. Glycan microarray analysis with a high infectivity "triple reassortant" virus ((A/Swine/MN/593/99 (H3N2)) that spread widely throughout the North American swine population and a lower infectivity human virus isolated from a single pig (A/Swine/ONT/00130/97 (H3N2)) showed that both viruses bound exclusively to glycans containing NeuAcα2-6, with strong binding to sialylated polylactosamine and sialylated N-glycans. Treatment with mannosamine precursors of sialic acid (to alter NeuAc/NeuGc abundances) and linkage-specific sialidases prior to infection indicated that the influenza viruses tested preferentially utilize NeuAcα2-6-sialylated glycans to infect SRECs. Our data indicate that NeuAcα2-6-terminated polylactosamine and sialylated N-glycans are important determinants for influenza viruses to infect SRECs. As NeuAcα2-6 polylactosamine glycans play major roles in human virus infection, the importance of these receptor components in virus infection of swine cells has implications for transmission of viruses between humans and pigs and for pigs as possible adaptation hosts of novel human influenza viruses.

DAS181 inhibits H5N1 influenza virus infection of human lung tissues.

DAS181 is a novel candidate therapeutic agent against influenza virus which functions via the mechanism of removing the virus receptor, sialic acid (Sia), from the adjacent glycan structures. DAS181 and its analogues have previously been shown to be potently active against multiple strains of seasonal and avian influenza virus strains in several experimental models, including cell lines, mice, and ferrets. Here we demonstrate that DAS181 treatment leads to desialylation of both alpha2-6-linked and alpha2-3-linked Sia in ex vivo human lung tissue culture and primary pneumocytes. DAS181 treatment also effectively protects human lung tissue and pneumocytes against the highly pathogenic avian influenza virus H5N1 (A/Vietnam/3046/2004). Two doses of DAS181 treatment given 12 h apart were sufficient to block H5N1 infection in the ex vivo lung tissue culture. These findings support the potential value of DAS181 as a broad-spectrum therapeutic agent against influenza viruses, especially H5N1.

Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays.

Plant lectin recognition of glycans was evaluated by SPR imaging using a model array of N-biotinylated aminoethyl glycosides of beta-D-glucose (negative control), alpha-D: -mannose (conA-responsive), beta-D-galactose (RCA(120)-responsive) and N-acetyl-beta-D-: glucosamine (WGA-responsive) printed onto neutravidin-coated gold chips. Selective recognition of the cognate ligand was observed when RCA(120) was passed over the array surface. Limited or no binding was observed for the non-cognate ligands. SPR imaging of an array of 40 sialylated and unsialylated glycans established the binding preference of hSiglec7 for alpha2-8-linked disialic acid structures over alpha2-6-sialyl-LacNAcs, which in turn were recognized and bound with greater affinity than alpha2-3-sialyl-LacNAcs. Affinity binding data could be obtained with as little as 10-20 microg of lectin per experiment. The SPR imaging technique was also able to establish selective binding to the preferred glycan ligand when analyzing crude culture supernatant containing 10-20 microg of recombinant hSiglec7-Fc. Our results show that SPR imaging provides results that are in agreement with those obtained from fluorescence based carbohydrate arrays but with the added advantage of label-free analysis.

Thioctic acid amides: convenient tethers for achieving low nonspecific protein binding to carbohydrates presented on gold surfaces.

The thioctic acid amides of 2'-aminoethyl alpha-d-mannopyranoside and 2'-aminoethyl alpha-1,3-d-mannopyranosyl (alpha-1,6-d-mannopyranosyl)-alpha-d-mannopyranoside presented on both planar and nanoparticle gold surfaces give higher specific and lower non-specific protein binding than the related 2'-thioethyl glycosides.