Ting-Jen R. Cheng

A consensus–hemagglutinin-based DNA vaccine that protects mice against divergent H5N1 influenza viruses

H5N1 influenza viruses have spread extensively among wild birds and domestic poultry. Cross-species transmission of these viruses to humans has been documented in over 380 cases, with a mortality rate of ≈60%. There is great concern that a H5N1 virus would acquire the ability to spread efficiently between humans, thereby becoming a pandemic threat. An H5N1 influenza vaccine must, therefore, be an integral part of any pandemic preparedness plan. However, traditional methods of making influenza vaccines have yet to produce a candidate that could induce potently neutralizing antibodies against divergent strains of H5N1 influenza viruses. To address this need, we generated a consensus H5N1 hemagglutinin (HA) sequence based on data available in early 2006. This sequence was then optimized for protein expression before being inserted into a DNA plasmid (pCHA5). Immunizing mice with pCHA5, delivered intramuscularly via electroporation, elicited antibodies that neutralized a panel of virions that have been pseudotyped with the HA from various H5N1 viruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4). Moreover, immunization with pCHA5 in mice conferred complete (clades 1 and 2.2) or significant (clade 2.1) protection from H5N1 virus challenges. We conclude that this vaccine, based on a consensus HA, could induce broad protection against divergent H5N1 influenza viruses and thus warrants further study.

Structure and bioactivity of the polysaccharides in medicinal plant Dendrobium huoshanense.

Detailed structures of the active polysaccharides extracted from the leaf and stem cell walls and mucilage of Dendrobium huoshanense are determined by using various techniques, including chromatographic, spectroscopic, chemical, and enzymatic methods. The mucilage polysaccharide exhibits specific functions in activating murine splenocytes to produce several cytokines including IFN-gamma, IL-10, IL-6, and IL-1alpha, as well as hematopoietic growth factors GM-CSF and G-CSF. However, the deacetylated mucilage obtained from an alkaline treatment fails to induce cytokine production. The structure and bioactivity of mucilage components are validated by further fractionation. This is the first study that provides clear evidence for the structure and activity relationship of the polysaccharide in D. huoshanense.

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.

Differential Receptor Binding Affinities of Influenza Hemagglutinins on Glycan Arrays

A library of 27 sialosides, including seventeen 2,3-linked and ten 2,6-linked glycans, has been prepared to construct a glycan array and used to profile the binding specificity of different influenza hemagglutinins (HA) subtypes, especially from the 2009 swine-originated H1N1 and seasonal influenza viruses. It was found that the HAs from the 2009 H1N1 and the seasonal Brisbane strain share similar binding profiles yet different binding affinities toward various α2,6 sialosides. Analysis of the binding profiles of different HA subtypes indicate that a minimum set of 5 oligosaccharides can be used to differentiate influenza H1, H3, H5, H7, and H9 subtypes. In addition, the glycan array was used to profile the binding pattern of different influenza viruses. It was found that most binding patterns of viruses and HA proteins are similar and that glycosylation at Asn27 is essential for receptor binding.

Domain requirement of moenomycin binding to bifunctional transglycosylases and development of high-throughput discovery of antibiotics

Moenomycin inhibits bacterial growth by blocking the transglycosylase activity of class A penicillin-binding proteins (PBPs), which are key enzymes in bacterial cell wall synthesis. We compared the binding affinities of moenomycin A with various truncated PBPs by using surface plasmon resonance analysis and found that the transmembrane domain is important for moenomycin binding. Full-length class A PBPs from 16 bacterial species were produced, and their binding activities showed a correlation with the antimicrobial activity of moenomycin against Enterococcus faecalis and Staphylococcus aureus. On the basis of these findings, a fluorescence anisotropy-based high-throughput assay was developed and used successfully for identification of transglycosylase inhibitors.

High-throughput identification of antibacterials against methicillin-resistant Staphylococcus aureus (MRSA) and the transglycosylase

To identify new transglycosylase inhibitors with potent anti-methicillin-resistant Staphylococcus aureus (MRSA) activities, a high-throughput screening against Staphylococcus aureus was conducted to look for antibacterial cores in our 2M compound library that consists of natural products, proprietary collection, and synthetic molecules. About 3600 hits were identified from the primary screening and the subsequent confirmation resulted in a total of 252 compounds in 84 clusters which showed anti-MRSA activities with MIC values as low as 0.1 μg/ml. Subsequent screening targeting bacterial transglycosylase identified a salicylanilide-based core that inhibited the lipid II polymerization and the moenomycin-binding activities of transglycosylase. Among the collected analogues, potent inhibitors with the IC(50) values below 10 μM against transglycosylase were identified. The non-carbonhydrate scaffold reported in this study suggests a new direction for development of bacterial transglycosylase inhibitors.

Crystal structure of Staphylococcus aureus transglycosylase in complex with a lipid II analog and elucidation of peptidoglycan synthesis mechanism

Bacterial transpeptidase and transglycosylase on the surface are essential for cell wall synthesis, and many antibiotics have been developed to target the transpeptidase; however, the problem of antibiotic resistance has arisen and caused a major threat in bacterial infection. The transglycosylase has been considered to be another excellent target, but no antibiotics have been developed to target this enzyme. Here, we determined the crystal structure of the Staphylococcus aureus membrane-bound transglycosylase, monofunctional glycosyltransferase, in complex with a lipid II analog to 2.3 Å resolution. Our results showed that the lipid II-contacting residues are not only conserved in WT and drug-resistant bacteria but also significant in enzymatic activity. Mechanistically, we proposed that K140 and R148 in the donor site, instead of the previously proposed E156, are used to stabilize the pyrophosphate-leaving group of lipid II, and E100 in the acceptor site acts as general base for the 4-OH of GlcNAc to facilitate the transglycosylation reaction. This mechanism, further supported by mutagenesis study and the structure of monofunctional glycosyltransferase in complex with moenomycin in the donor site, provides a direction for antibacterial drugs design.

Synthesis and Evaluation of a New Fluorescent Transglycosylase Substrate: Lipid II-Based Molecule Possessing a Dansyl-C20 Polyprenyl Moiety

The preparation of a novel fluorescent lipid II-based substrate for transglycosylases (TGases) is described. This substrate has characteristic structural features including a shorter lipid chain, a fluorophore tag at the end of the lipid chain rather than on the peptide chain, and no labeling with a radioactive atom. This fluorescent substrate is readily utilized in TGase activity assays to characterize TGases and also to evaluate the activities of TGase inhibitors.

Synthesis of Neisseria meningitidis Serogroup W135 Capsular Oligosaccharides for Immunogenicity Comparison and Vaccine Development

Carbohydrate VaccineC.-H. Wang, S.-T. Li, T.-L. Lin, Y.-Y. Cheng,T.-H. Sun, J.-T. Wang, T.-J. R. Cheng,K. K. T. Mong, C.-H. Wong,C.-Y. Wu* &&&&—&&&&Synthesis of Neisseria meningitidisSerogroup W135 CapsularOligosaccharides for ImmunogenicityComparison and Vaccine DevelopmentSweetening the deal: N. meningitidisserogroup W135 capsular oligosacchar-ides were synthesized in lengths fromdisaccharides to decasaccharides. Serafrom mice immunized with these oligo-saccharide–protein conjugates wereexamined by a glycan microarray (seepicture) and bactericidal assay for anti-body specificity and the ability to killbacteria.