Chung-Yi Wu

Glycans on influenza hemagglutinin affect receptor binding and immune response

Recent cases of avian influenza H5N1 and the swine-origin 2009 H1N1 have caused a great concern that a global disaster like the 1918 influenza pandemic may occur again. Viral transmission begins with a critical interaction between hemagglutinin (HA) glycoprotein, which is on the viral coat of influenza, and sialic acid (SA) containing glycans, which are on the host cell surface. To elucidate the role of HA glycosylation in this important interaction, various defined HA glycoforms were prepared, and their binding affinity and specificity were studied by using a synthetic SA microarray. Truncation of the N-glycan structures on HA increased SA binding affinities while decreasing specificity toward disparate SA ligands. The contribution of each monosaccharide and sulfate group within SA ligand structures to HA binding energy was quantitatively dissected. It was found that the sulfate group adds nearly 100-fold (2.04 kcal/mol) in binding energy to fully glycosylated HA, and so does the biantennary glycan to the monoglycosylated HA glycoform. Antibodies raised against HA protein bearing only a single N-linked GlcNAc at each glycosylation site showed better binding affinity and neutralization activity against influenza subtypes than the fully glycosylated HAs elicited. Thus, removal of structurally nonessential glycans on viral surface glycoproteins may be a very effective and general approach for vaccine design against influenza and other human viruses.

Broadly Neutralizing HIV Antibodies Define a Glycan-Dependent Epitope on the Prefusion Conformation of gp41 on Cleaved Envelope Trimers

Broadly neutralizing HIV antibodies are much sought after (a) to guide vaccine design, both as templates and as indicators of the authenticity of vaccine candidates, (b) to assist in structural studies, and (c) to serve as potential therapeutics. However, the number of targets on the viral envelope spike for such antibodies has been limited. Here, we describe a set of human monoclonal antibodies that define what is, to the best of our knowledge, a previously undefined target on HIV Env. The antibodies recognize a glycan-dependent epitope on the prefusion conformation of gp41 and unambiguously distinguish cleaved from uncleaved Env trimers, an important property given increasing evidence that cleavage is required for vaccine candidates that seek to mimic the functional HIV envelope spike. The availability of this set of antibodies expands the number of vaccine targets on HIV and provides reagents to characterize the native envelope spike.

Toward Automated Oligosaccharide Synthesis

Carbohydrates have been shown to play important roles in biological processes. The pace of development in carbohydrate research is, however, relatively slow due to the problems associated with the complexity of carbohydrate structures and the lack of general synthetic methods and tools available for the study of this class of biomolecules. Recent advances in synthesis have demonstrated that many of these problems can be circumvented. In this Review, we describe the methods developed to tackle the problems of carbohydrate-mediated biological processes, with particular focus on the issue related to the development of the automated synthesis of oligosaccharides. Further applications of carbohydrate microarrays and vaccines to human diseases are also highlighted.

Hemagglutinin–neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets

A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin–neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility.

Glycan arrays: biological and medical applications

Carbohydrates and their conjugates are involved in various biological events, including viral and bacterial infection, the immune response, differentiation and development, and the progression of tumor cell metastasis. Glycan arrays are a new technology that has enabled the high-sensitivity and rapid analysis carbohydrate–protein interaction and contribute to significant advances in glycomics. Glycan arrays use a minute amount of materials and can be used for high-throughput profiling and quantitative analysis and provide information for the development of carbohydrate-based vaccines and new drug discovery.

Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer

Cancer-associated carbohydrate antigens are often found on the surface of cancer cells. Understanding their roles in cancer progression will lead to the development of new therapeutics and high-sensitivity diagnostics for cancers. Globo H is a member of this family, which is highly expressed on breast cancer cells. Here, we report the development of a glycan microarray of Globo H and its analogs for measurement of the dissociation constants on surface (KD,surf) with three different monoclonal antibodies (VK-9, Mbr1, and anti-SSEA-3), to deduce their binding specificity. The glycan microarray was also used to detect the amount of antibodies present in the plasma of breast cancer patients and normal blood donors. It was shown that the amount of antibodies against Globo H from breast cancer patients were significantly higher than normal blood donors, providing a new tool for possible breast cancer diagnosis. Compared with the traditional ELISA method, this array method required only atto-mole amounts of materials and is more effective and more sensitive (5 orders of magnitude). The glycan microarray thus provides a new platform for use to monitor the immune response to carbohydrate epitopes after vaccine therapy or during the course of cancer progression.

Highly Alpha‐Selective Sialyl Phosphate Donors for Efficient Preparation of Natural Sialosides

N-Acetyl neuraminic acid (Neu5Ac) is most frequently found at the terminal end of glycoconjugates on the cell surface. This terminally exposed position allows Neu5Ac-containing conjugates to be exploited as receptors for viruses and bacteria, in addition to governing a wide variety of biological processes, such as tumor metastasis, cell differentiation, and cell–cell interactions. In naturally occurring sialosides, Neu5Ac is found linked to galactosides through the aACHTUNGTRENNUNG(2!3) or aACHTUNGTRENNUNG(2!6) linkage in Nand O-linked glycoproteins, and also to N-acetylgalactosamine through the aACHTUNGTRENNUNG(2!6) linkage in O-linked glycoproteins. In addition, polysialosides formed by the aACHTUNGTRENNUNG(2!8) or a ACHTUNGTRENNUNG(2!9) linkages are constituents of glycoproteins and glycolipids. The biological significance of sialoside receptors has driven research for their efficient synthesis. This has included significant efforts directed toward the development of sialic acid donors for efficient a-sialylation, including the use of anomeric leaving groups, such as halides, phosphites, sulfides, xanthates, and phenyltrifluoroacetimidates, the introduction of an auxiliary group at C-1 and C-3, the modification of the Nacetyl functional group at C-5, or the optimized combinations of the leaving group with positional modification. However, high yielding a-selective sialylation is still problematic owing to the presence of the C-1 electron-withdrawing carboxyl group at the tertiary anomeric center and the lack of a participating group at C-3 to direct the stereochemical outcome of glycosylation. The development of new strategies for the convergent synthesis of saccharides is a major focus in carbohydrate chemistry. Approaches such as armed–disarmed; onepot; reactivity-based, programmable one-pot; solidphase; orthogonal; and pre-activation methods have been developed to improve efficiency, with the ultimate goal of developing an automated method for oligosaccharide synthesis. The basic concept of these strategies involves the selective activation of one donor in the presence of an acceptor with the same or different leaving groups, so the newly formed product can be directly elongated without further aglycon leaving group adjustment. However, these approaches are not applicable to all glycosides. For example, sialic acid thioglycosides have limitations in our programmable reactivity-based, one-pot strategy due to their poor and narrow range of anomeric reactivity values (RRVs). To resolve the problem, sialylated disaccharides have been used as building blocks. However, application of this strategy is limited by the lack of an efficient and a-selective sialic acid donor that possesses a leaving group orthogonal to the thioglycoside. Herein, we report a new sialylation reagent 1 that employs an N-acetyl-5-N,4-O-carbonyl protection with dibutyl phosphate as the leaving group. Furthermore, the new donor was applied to develop efficient strategies to use to create natural sialosides. Many researchers have focused on the modification of the N-acetyl functional group of sialic acid for highly efficient a-sialylation. However, there is no general donor for different acceptors. The choice of C-5 modification to be used is highly dependent on protecting groups and the nature of glycosyl acceptors. There is an excellent review on C-5 modification by De Meo. Crich and co-workers have shown that the N-acetyl-5-N,4-O-carbonyl thiosialoside can [a] C.-H. Hsu, Dr. K.-C. Chu, Y.-S. Lin, Dr. J.-L. Han, Y.-S. Peng, Dr. C.-T. Ren, Prof. C.-Y. Wu, Prof. C.-H. Wong The Genomics Research Center, Academia Sinica (Taiwan) 128 Academia Road, Section 2, Nankang, Taipei 115 (Taiwan) Fax: (+886)2-2785-3852 E-mail : cyiwu@gate.sinica.edu.tw chwong@gate.sinica.edu.tw [b] C.-H. Hsu, Prof. C.-H. Wong Department of Chemistry, The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA) [c] C.-H. Hsu, Prof. C.-Y. Wu, Prof. C.-H. Wong Chemical Biology and Molecular Biophysics Taiwan International Graduate Program, Academia Sinica 128 Academia Road, Section 2, Nankang, Taipei 115 (Taiwan) [d] C.-H. Hsu Institute of Bioinformatics and Structural Biology National Tsing-Hua University, Hsin-Chu (Taiwan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200903035.

High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity

As influenza viruses have developed resistance towards current drugs, new inhibitors that prevent viral replication through different inhibitory mechanisms are useful. In this study, we developed a screening procedure to search for new antiinfluenza inhibitors from 1,200,000 compounds and identified previously reported as well as new antiinfluenza compounds. Several antiinfluenza compounds were inhibitory to the influenza RNA-dependent RNA polymerase (RdRP), including nucleozin and its analogs. The most potent nucleozin analog, 3061 (FA-2), inhibited the replication of the influenza A/WSN/33 (H1N1) virus in MDCK cells at submicromolar concentrations and protected the lethal H1N1 infection of mice. Influenza variants resistant to 3061 (FA-2) were isolated and shown to have the mutation on nucleoprotein (NP) that is distinct from the recently reported resistant mutation of Y289H [Kao R, et al. (2010) Nat Biotechnol 28:600]. Recombinant influenza carrying the Y52H NP is also resistant to 3061 (FA-2), and NP aggregation induced by 3061 (FA-2) was identified as the most likely cause for inhibition. In addition, we identified another antiinfluenza RdRP inhibitor 367 which targets PB1 protein but not NP. A mutant resistant to 367 has H456P mutation at the PB1 protein and both the recombinant influenza and the RdRP expressing the PB1 H456P mutation have elevated resistance to 367. Our high-throughput screening (HTS) campaign thus resulted in the identification of antiinfluenza compounds targeting RdRP activity.

Carbohydrate-based vaccines with a glycolipid adjuvant for breast cancer

Globo H (GH) is a hexasaccharide specifically overexpressed on a variety of cancer cells and therefore, a good candidate for cancer vaccine development. To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked GH to a carrier protein, including keyhole limpet hemocyanion, diphtheria toxoid cross-reactive material (CRM) 197 (DT), tetanus toxoid, and BSA, and combined with an adjuvant, and it was administered to mice for the study of immune response. Glycan microarray analysis of the antiserum obtained indicated that the combination of GH-DT adjuvanted with the α-galactosylceramide C34 has the highest enhancement of anti-GH IgG. Compared with the phase III clinical trial vaccine, GH–keyhole limpet hemocyanion/QS21, the GH-DT/C34 vaccine elicited more IgG antibodies, which are more selective for GH and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) and SSEA4, all of which were specifically overexpressed on breast cancer cells and breast cancer stem cells with SSEA4 at the highest level (>90%). We, therefore, further developed SSEA4-DT/C34 as a vaccine candidate, and after immunization, it was found that the elicited antibodies are also IgG-dominant and very specific for SSEA4.

Iron Oxide/Gold Core/Shell Nanoparticles for Ultrasensitive Detection of Carbohydrate−Protein Interactions

Changes in the expression of cell surface glycan are often associated with malignant metastasis. The expression level may be dramatically enhanced during tumor progression. A highly sensitive assay that is capable of detecting low levels of cancer-associated carbohydrate antigens can be a powerful tool for early diagnosis. In this work, an ultrasensitive glycans array using iron oxide/gold core/shell nanoparticles conjugated with antibodies or proteins is developed. A magnetic field is applied to quickly bring nanoparticle labeled proteins or antibodies from a solution to an array of carbohydrates immobilized on glass slides and to help them to encounter the carbohydrates at very low concentration. The gold shell provides a well established platform for conjugation of biomolecules. Well-defined recognition systems, namely, mannose derivatives (Man1, Man4, and Man9) with a mannose binding lectin (Concanavalin A) and a stage-specific embryonic antigens-3 (SSEA-3) with a monoclonal antibody (anti-SSEA-3) were chosen to establish this detection tool. Array systems were conducted to determine their surface dissociation constant (K(D,surface)) and their binding specificity for qualitative and quantitative analysis of carbohydrate-protein and carbohydrate-antibody interactions. When coupled with a signal amplification method based on nanoparticle-promoted reduction of silver, the sensitivity of an iron oxide/gold core/shell nanoparticle-based assay reached to subattomole level in carbohydrate detection.