Pseudaminic Acid on Exopolysaccharide of Acinetobacter baumannii Plays a Critical Role in Phage-Assisted Preparation of Glycoconjugate Vaccine with High Antigenicity.

Pseudaminic acid (Pse) has been known for participating in crucial bacterial virulence and thus is an attractive target in the development of glycoconjugate vaccine. Particularly, this therapeutic alternative was suggested to be a potential solution against antibiotic resistant Acinetobacter baumannii that poses a serious global health threat. Also, Pse was found to be involved in the exopolysaccharide (EPS) of mild antibiotic resistant A. baumannii strain 54149 ( Ab-54149) of which specific glycosyl linkage can be depolymerized by phage ΦAB6 tailspike protein (ΦAB6TSP). In this study, we found that the antibodies induced by Ab-54149 EPS was capable of recognizing a range of EPS of other clinical A. baumannii strains, and deemed as a great potential material for vaccination. To efficiently acquire homogeneous EPS-derived oligosaccharide with significant immunogenic activity for the production of glycoconjugate, we used the ΦAB6TSP for the fragmentation of Ab-54149 EPS instead of chemical methods. Moreover, insight into the ligand binding characterization of ΦAB6TSP suggested the branched Pse on the Ab-54149 EPS served as a recognition site of ΦAB6TSP. The serum boosted by ΦAB6TSP-digested product and carrier protein CRM197 conjugate complex displayed specific sensitivity toward Ab-54149 EPS with bacterial killing activity. Strikingly, Pse is an ideal epitope with strong antigenicity, profiting the application of the probe for pathogen detection and glyco-based vaccine.

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.

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes.

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galß1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.

Immunogenicity study of Globo H analogues with modification at the reducing or nonreducing end of the tumor antigen.

Globo H-based therapeutic cancer vaccines have been tested in clinical trials for the treatment of late stage breast, ovarian, and prostate cancers. In this study, we explored Globo H analogue antigens with an attempt to enhance the antigenic properties in vaccine design. The Globo H analogues with modification at the reducing or nonreducing end were synthesized using chemoenzymatic methods, and these modified Globo H antigens were then conjugated with the carrier protein diphtheria toxoid cross-reactive material (CRM) 197 (DT), and combined with a glycolipid C34 as an adjuvant designed to induce a class switch to form the vaccine candidates. After Balb/c mice injection, the immune response was studied by a glycan array and the results showed that modification at the C-6 position of reducing end glucose of Globo H with the fluoro, azido, or phenyl group elicited IgG antibody response to specifically recognize Globo H (GH) and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) (also called Gb5) and stage-specific embryonic antigen 4 (SSEA4). However, only the modification of Globo H with the azido group at the C-6 position of the nonreducing end fucose could elicit a strong IgG immune response. Moreover, the antibodies induced by these vaccines were shown to recognize GH expressing tumor cells (MCF-7) and mediate the complement-dependent cell cytotoxicity against tumor cells. Our data suggest a new potential approach to cancer vaccine development.

Broadly neutralizing DNA vaccine with specific mutation alters the antigenicity and sugar-binding activities of influenza hemagglutinin.

The rapid genetic drift of influenza virus hemagglutinin is an obstacle to vaccine efficacy. Previously, we found that the consensus hemagglutinin DNA vaccine (pCHA5) can only elicit moderate neutralization activities toward the H5N1 clade 2.1 and clade 2.3 viruses. Two approaches were thus taken to improve the protection broadness of CHA5. The first one was to include certain surface amino acids that are characteristic of clade 2.3 viruses to improve the protection profiles. When we immunized mice with CHA5 harboring individual mutations, the antibodies elicited by CHA5 containing P157S elicited higher neutralizing activity against the clade 2.3 viruses. Likewise, the viruses pseudotyped with hemagglutinin containing 157S became more susceptible to neutralization. The second approach was to update the consensus sequence with more recent H5N1 strains, generating a second-generation DNA vaccine pCHA5II. We showed that pCHA5II was able to elicit higher cross-neutralization activities against all H5N1 viruses. Comparison of the neutralization profiles of CHA5 and CHA5II, and the animal challenge studies, revealed that CHA5II induced the broadest protection profile. We concluded that CHA5II combined with electroporation delivery is a promising strategy to induce antibodies with broad cross-reactivities against divergent H5N1 influenza viruses.

Efficient convergent synthesis of bi-, tri-, and tetra-antennary complex type N-glycans and their HIV-1 antigenicity.

The structural diversity of glycoproteins often comes from post-translational glycosylation with heterogeneous N-glycans. Understanding the complexity of glycans related to various biochemical processes demands a well-defined synthetic sugar library. We report herein a unified convergent strategy for the rapid production of bi-, tri-, and tetra-antennary complex type N-glycans with and without terminal N-acetylneuraminic acid residues connected via the α-2,6 or α-2,3 linkages. Moreover, using sialyltransferases to install sialic acid can minimize synthetic steps through the use of shared intermediates to simplify the complicated procedures associated with conventional sialic acid chemistry. Furthermore, these synthetic complex oligosaccharides were compiled to create a glycan array for the profiling of HIV-1 broadly neutralizing antibodies PG9 and PG16 that were isolated from HIV infected donors. From the study of antibody PG16, we identified potential natural and unnatural glycan ligands, which may facilitate the design of carbohydrate-based immunogens and hasten the HIV vaccine development.

Synthesis and vaccine evaluation of the tumor-associated carbohydrate antigen RM2 from prostate cancer.

We have successfully developed a [1+2+3] one-pot strategy to synthesize the RM2 antigen hexasaccharide that was proposed to be a prostate tumor antigen. The structure of the synthetic product was verified by NMR analysis and antibody binding assay using a glycan microarray. In addition, the synthetic antigen was conjugated to a mutated diphtheria toxin (DT, CRM197) with different copy numbers and adjuvant combinations to form the vaccine candidates. After vaccination in mice, we used glycan microarrays to monitor their immune response, and the results indicated that, when one molecule of DT was incorporated with 4.7 molecules of RM2 on average (DT-RM4.7) and adjuvanted with the glycolipid C34, the combination exhibited the strongest anti-RM2 IgG titer. Moreover, the induced mouse antibodies mediated effective complement-dependent cytotoxicity (CDC) against the prostate cancer cell line LNCap.

Chemical probes for drug-resistance assessment by binding competition (RABC): oseltamivir susceptibility evaluation.

The wizard of OS (resistance): the binding difference of neuraminidase inhibitors (zanamivir versus oseltamivir (OS)) was used to establish an assay to identify the influenza subtypes that are resistant to OS but still sensitive to zanamivir. This assay used a zanamivir-biotin conjugate to determine the OS susceptibility of a wide range of influenza viruses and over 200 clinical isolates.

Ganglioside-focused Glycan Array Reveals Abnormal Anti-GD1b Auto-antibody in Plasma of Preclinical Huntington's Disease.

Huntington's disease (HD) is a progressive and devastating neurodegenerative disease marked by inheritable CAG nucleotide expansion. For offspring of HD patients carrying abnormal CAG expansion, biomarkers that predict disease onset are crucially important but still lacking. Alteration of brain ganglioside patterns has been observed in the pathology of patients carrying HD. Here, by using a novel and sensitive ganglioside-focused glycan array, we examined the potential of anti-glycan auto-antibodies for HD. In this study, we collected plasma from 97 participants including 42 control (NC), 16 pre-manifest HD (pre-HD), and 39 HD cases and measured the anti-glycan auto-antibodies by a novel ganglioside-focused glycan array. The association between plasma anti-glycan auto-antibodies and disease progression was analyzed using univariate and multivariate logistic regression. The disease-predictive capacity of anti-glycan auto-antibodies was further investigated by receiver operating characteristic (ROC) analysis. We found that anti-glycan auto-antibodies were generally higher in the pre-HD group when compared to the NC and HD groups. Specifically, anti-GD1b auto-antibody demonstrated the potential for distinguishing between pre-HD and control groups. Moreover, in combination with age and the number of CAG repeat, the level of anti-GD1b antibody showed excellent predictability with an area under the ROC curve (AUC) of 0.95 to discriminate between pre-HD carriers and HD patients. With glycan array technology, this study demonstrated abnormal auto-antibody responses that showed temporal changes from pre-HD to HD.

Synthesis of Azido-Globo H Analogs for Immunogenicity Evaluation.

Globo H (GH) is a tumor-associated carbohydrate antigen (TACA), and GH conjugations have been evaluated as potential cancer vaccines. However, like all carbohydrate-based vaccines, low immunogenicity is a major issue. Modifications of the TACA increase its immunogenicity, but the systemic modification on GH is challenging and the synthesis is cumbersome. In this study, we synthesized several azido-GH analogs for evaluation, using galactose oxidase to selectively oxidize C6-OH of the terminal galactose or N-acetylgalactosamine on lactose, Gb3, Gb4, and SSEA3 into C6 aldehyde, which was then transformed chemically to the azido group. The azido-derivatives were further glycosylated to azido-GH analogs by glycosyltransferases coupled with sugar nucleotide regeneration. These azido-GH analogs and native GH were conjugated to diphtheria toxoid cross-reactive material CRM197 for vaccination with C34 adjuvant in mice. Glycan array analysis of antisera indicated that the azido-GH glycoconjugate with azide at Gal-C6 of Lac (1-CRM197) elicited the highest antibody response not only to GH, SSEA3, and SSEA4, which share the common SSEA3 epitope, but also to MCF-7 cancer cells, which express these Globo-series glycans.

Development of Klebsiella pneumoniae Capsule Polysaccharide-Conjugated Vaccine Candidates Using Phage Depolymerases.

Klebsiella pneumoniae is an important pathogen associated with nosocomial infection and has developed increasing resistance to antibiotics such as extended-spectrum ß-lactams and carbapenem. In recent years, K. pneumoniae isolates have emerged as a major cause of global community-acquired infections such as pneumonia and pyogenic liver abscess. Although serotypes K1 and K2 have been identified as the predominant capsular types associated with invasive infections, no K. pneumoniae vaccine is commercially available, probably due to immunogenicity loss in the traditional depolymerization method to obtain capsule polysaccharide (CPS) for the preparation of conjugated vaccine. In this study, we successfully retained immunogenicity by using K1 (K1-ORF34) and K2 (K2-ORF16) CPS depolymerases that were identified from phages to cleave K1 and K2 CPSs into intact structural units of oligosaccharides with intact modifications. The obtained K1 and K2 oligosaccharides were separately conjugated with CRM197 carrier protein to generate CPS-conjugated vaccines. Immunization experiments of mice showed both K1 and K2 CPS-conjugated vaccines induced anti-CPS antibodies with 128-fold and 64-fold increases of bactericidal activities, respectively, compare to mice without vaccinations. Challenge tests indicated that K1 or K2 CPS-conjugated vaccine and divalent vaccine (a mixture of K1 and K2 CPS-conjugated vaccines) protected mice from subsequent infection of K. pneumoniae by the respective capsular type. Thus, we demonstrated K1 and K2 CPS-conjugated vaccines prepared by CPS depolymerases is a promising candidate for developing vaccines against human K. pneumoniae infections.

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 (K(D,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.

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.

Enhancement of fucosylated N-glycan isomer separation with an ultrahigh column temperature in porous graphitic carbon liquid chromatography-mass spectrometry.

Due to the heterogeneous and isomeric nature of glycans, the development of an advanced separation of distinct glycan isomers is essential for glycan research and application. In this study, we utilized porous graphite carbon (PGC) chromatography for the separation of isomeric oligosaccharides without reduction or chemical derivatization at 190 °C in a custom-built heating oven. Furthermore, the fine structures of glycan isomers could be identified by using ultrahigh temperature PGC liquid chromatography mass spectrometry (UHT-PGC-LCMS). A nonreduced hydrolyzed dextran was applied to verify the performance of UHT-PGC. When the temperature of the PGC column was increased from 25 to 190 °C, the liquid chromatography separation power of the nonreduced dextran ladder significantly increased. The advantage of the UHT-PGC column was its high peak capacity with gradient elution in 10 min at 190 °C, 6700 psi, and a 250 µL/min flow rate for native glycan analysis. Four synthetic Lewis antigen isomers were used to elucidate the separation effectiveness in UHT-PGC. Moreover, mass spectrometry-based sequencing to generate specific diagnostic ions from the four synthetic Lewis antigens was used to predict isomeric glycans based on the relative intensity ratio (RIR) of diagnostic ions. The intensities of the diagnostic ions of synthetic isomers were used to identify each isomer of the fucosylated glycan. The results clearly showed that terminal Lewis A and X residues were in the 3- and 6-arms of N-glycan, respectively.

Synthesis of Asymmetric N-Glycans as Common Core Substrates for Structural Diversification through Selective Enzymatic Glycosylation.

N-glycans on the cell surface provide distinct signatures that are recognized by different glycan-binding proteins (GBPs) and pathogens. Most glycans in humans are asymmetric and isomeric, yet their biological functions are not well understood due to their lack of availability for studies. In this work, we have developed an improved strategy for asymmetric N-glycan assembly and diversification using designed common core substrates prepared chemically for selective enzymatic fucosylation and sialylation. The resulting 26 well-defined glycans that carry the sialic acid residue on different antennae were used in a microarray as a representative application to profile the binding specificity of hemagglutinin (HA) from the avian influenza virus (H5N2). We found distinct binding affinity for the Neu5Ac-Gal epitope linked to the N-acetylglucosamine (GlcNAc) of different branches and only a minor effect in binding for the terminal galactose on different branches. Overall, the microarray analysis showed branch-biased and context-based recognition patterns.

HA-pseudotyped retroviral vectors for influenza antagonist screening.

Influenza infections are initiated by the binding of the influenza hemagglutinin (HA) and the cellular receptor sialic acids. The binding is followed by internalization, endocytosis, and uncoating to release the influenza genome to the cytoplasm. It is conceivable that specific inhibitors that antagonize any one of these events could prevent the replication of influenza infections. The authors made HA pseudotyped retroviral vectors that express luciferase reporter activities upon transduction to several recipient cells. The transduction of the HA-pseudotype virus particles (HApp) was mediated through the specific interactions between an avian HA and the terminal disaccharides of sialic acid (SA) and galactose (Gal) in alpha-2,3 linkage. The HApp-mediated transduction method was used to develop a high-throughput screening assay and to screen for hits from a fermentation extract library. Specific hits that inhibited the HA-mediated but were noninhibitory to the vesicular stomatitis virus-mediated pseudoviral transductions were identified. A few of these hits have anti-influenza activities that prevent the replication of both H1N1 (WSN) and H5N1 (RG14) influenza viruses.

Desorption ionization of biomolecules on metals.

Direct desorption ionization of various types of biomolecules on metal substrates without the need of matrices was observed by a time-of-flight mass spectrometer. It provides a new convenient method for detection of small biomolecules without the confusion of ion peaks from matrix compounds. Simple commercial Al foil can be used as the substrate to obtain mass spectra of biomolecules without the need of an etching process to produce a porous surface such as with direct ionization on silicon (DIOS). The desorption and ionization mechanism is also discussed.

An Alkynyl-Fucose Halts Hepatoma Cell Migration and Invasion by Inhibiting GDP-Fucose-Synthesizing Enzyme FX, TSTA3.

Fucosylation is a glycan modification critically involved in cancer and inflammation. Although potent fucosylation inhibitors are useful for basic and clinical research, only a few inhibitors have been developed. Here, we focus on a fucose analog with an alkyne group, 6-alkynyl-fucose (6-Alk-Fuc), which is used widely as a detection probe for fucosylated glycans, but is also suggested for use as a fucosylation inhibitor. Our glycan analysis using lectin and mass spectrometry demonstrated that 6-Alk-Fuc is a potent and general inhibitor of cellular fucosylation, with much higher potency than the existing inhibitor, 2-fluoro-fucose (2-F-Fuc). The action mechanism was shown to deplete cellular GDP-Fuc, and the direct target of 6-Alk-Fuc is FX (encoded by TSTA3), the bifunctional GDP-Fuc synthase. We also show that 6-Alk-Fuc halts hepatoma invasion. These results highlight the unappreciated role of 6-Alk-Fuc as a fucosylation inhibitor and its potential use for basic and clinical science.

Structural basis for fragmenting the exopolysaccharide of Acinetobacter baumannii by bacteriophage ΦAB6 tailspike protein.

With an increase in antibiotic-resistant strains, the nosocomial pathogen Acinetobacter baumannii has become a serious threat to global health. Glycoconjugate vaccines containing fragments of bacterial exopolysaccharide (EPS) are an emerging therapeutic to combat bacterial infection. Herein, we characterize the bacteriophage ΦAB6 tailspike protein (TSP), which specifically hydrolyzed the EPS of A. baumannii strain 54149 (Ab-54149). Ab-54149 EPS exhibited the same chemical structure as two antibiotic-resistant A. baumannii strains. The ΦAB6 TSP-digested products comprised oligosaccharides of two repeat units, typically with stoichiometric pseudaminic acid (Pse). The 1.48-1.89-Å resolution crystal structures of an N-terminally-truncated ΦAB6 TSP and its complexes with the semi-hydrolyzed products revealed a trimeric ß-helix architecture that bears intersubunit carbohydrate-binding grooves, with some features unusual to the TSP family. The structures suggest that Pse in the substrate is an important recognition site for ΦAB6 TSP. A region in the carbohydrate-binding groove is identified as the determinant of product specificity. The structures also elucidated a retaining mechanism, for which the catalytic residues were verified by site-directed mutagenesis. Our findings provide a structural basis for engineering the enzyme to produce desired oligosaccharides, which is useful for the development of glycoconjugate vaccines against A. baumannii infection.