An intranasal cationic liposomal polysaccharide vaccine elicits humoral immune responses against Streptococcus pneumoniae.

Diseases caused by S. pneumoniae are the leading cause of child mortality. As antibiotic resistance of S. pneumoniae is rising, vaccination remains the most recommended solution. However, the existing pneumococcal polysaccharides vaccine (Pneumovax® 23) proved only to induce T-independent immunity, and strict cold chain dependence of the protein conjugate vaccine impedes its promotion in developing countries, where infections are most problematic. Affordable and efficient vaccines against pneumococcus are therefore in high demand. Here, we present an intranasal vaccine Lipo+CPS12F&αGC, containing the capsular polysaccharides of S. pneumoniae 12F and the iNKT agonist α-galactosylceramide in cationic liposomes. In BALB/cJRj mice, the vaccine effectively activates iNKT cells and promotes B cells maturation, stimulates affinity-matured IgA and IgG production in both the respiratory tract and systemic blood, and displays sufficient protection both in vivo and in vitro. The designed vaccine is a promising, cost-effective solution against pneumococcus, which can be expanded to cover more serotypes and pathogens.

Chemical synthesis of natural and azido-modified UDP-rhamnose and -arabinofuranose for glycan array-based characterization of plant glycosyltransferases.

Chemical syntheses of UDP-rhamnose and UDP-arabinofuranose and respective azido-modified analogues are reported. The prepared substrates are useful for the glycan array-based analysis of glycosyltransferases, as exemplified with the plant cell wall-biosynthetic enzymes PvXAT3, AtRRT4 and PtRRT5.

Chemical synthesis and immunological evaluation of cancer vaccines based on ganglioside antigens and α-galactosylceramide.

iNKT cells - often referred as the "Swiss Army knife" of the immune system - have emerged as central players in cancer vaccine therapies. Glycolipids activating iNKT cells, such as α-galactosylceramide (αGalCer), can enhance the immune response against co-delivered cancer antigens and have been applied in the design of self-adjuvanting anti-tumor vaccines. In this context, this work focuses on the chemical synthesis of ganglioside tumor-associated carbohydrate antigens (TACAs), namely GM3 and (Neu5Gc)GM3 antigens, their conjugation to αGalCer, and their formulation into liposomes as an efficient platform for their in vivo delivery. Liposomes containing GM3-αGalCer, (Neu5Gc)GM3-αGalCer, and equimolar amounts of the two conjugates have been fully characterized and their ability to activate iNKT cell has been confirmed ex vivo in mouse and human cell assays. The candidates were tested in in vivo immunization studies, demonstrating an ability to induce both TH1 and TH2 cytokines leading to the production of all subclasses of IgG antibodies. Notably, the study also demonstrated that serum antibodies raised against the two TACAs, alone and in combination, were cross-reactive. This finding has consequences for future vaccine designs - even if a highly tumor-selective antigen is chosen, the resulting antibody response may be broader than anticipated.

Synthesis of fluoro- and seleno-containing D-lactose and D-galactose analogues.

Synthetic deoxy-fluoro-carbohydrate derivatives and seleno-sugars are useful tools in protein-carbohydrate interaction studies using nuclear magnetic resonance spectroscopy because of the presence of the 19F and 77Se reporter nuclei. Seven saccharides containing both these atoms have been synthesized, three monosaccharides, methyl 6-deoxy-6-fluoro-1-seleno-ß-D-galactopyranoside (1) and methyl 2-deoxy-2-fluoro-1-seleno-α/ß-D-galactopyranoside (2α and 2ß), and four disaccharides, methyl 4-O-(ß-D-galactopyranosyl)-2-deoxy-2-fluoro-1-seleno-ß-D-glucopyranoside (3), methyl 4-Se-(ß-D-galactopyranosyl)-2-deoxy-2-fluoro-4-seleno-ß-D-glucopyranoside (4), and methyl 4-Se-(2-deoxy-2-fluoro-α/ß-D-galactopyranosyl)-4-seleno-ß-D-glucopyranoside (5α and 5ß), the three latter compounds with an interglycosidic selenium atom. Selenoglycosides 1 and 3 were obtained from the corresponding bromo sugar by treatment with dimethyl selenide and a reducing agent, while compounds 2α/2ß, 4, and 5α/5ß were synthesized by the coupling of a D-galactosyl selenolate, obtained in situ from the corresponding isoselenouronium salt, with either methyl iodide or a 4-O-trifluoromethanesulfonyl D-galactosyl moiety. While benzyl ether protecting groups were found to be incompatible with the selenide linkage during deprotection, a change to acetyl esters afforded 4 in a 17% overall yield and over 9 steps from peracetylated D-galactosyl bromide. The synthesis of 5 was performed similarly, but the 2-fluoro substituent led to reduced stereoselectivity in the formation of the isoselenouronium salt (α/ß âˆ¼ 1 : 2.3). However, the ß-anomer of the uronium salt could be obtained almost pure (∼98%) by precipitation from the reaction mixture. The following displacement reaction occurred without anomerisation, affording, after deacetylation, pure 5ß.

Rhamnogalacturonan II: Chemical Synthesis of a Substructure Including α-2,3-Linked Kdo*.

The synthesis of a fully deprotected Kdo-containing rhamnogalacturonan II pentasaccharide is described. The strategy relies on the preparation of a suitably protected homogalacturonan tetrasaccharide backbone, through a post-glycosylation oxidation approach, and its stereoselective glycosylation with a Kdo fluoride donor.

Fluorinated Carbohydrates as Lectin Ligands: Simultaneous Screening of a Monosaccharide Library and Chemical Mapping by 19F NMR Spectroscopy.

Molecular recognition of carbohydrates is a key step in essential biological processes. Carbohydrate receptors can distinguish monosaccharides even if they only differ in a single aspect of the orientation of the hydroxyl groups or harbor subtle chemical modifications. Hydroxyl-by-fluorine substitution has proven its merits for chemically mapping the importance of hydroxyl groups in carbohydrate-receptor interactions. 19F NMR spectroscopy could thus be adapted to allow contact mapping together with screening in compound mixtures. Using a library of fluorinated glucose (Glc), mannose (Man), and galactose (Gal) derived by systematically exchanging every hydroxyl group by a fluorine atom, we developed a strategy combining chemical mapping and 19F NMR T2 filtering-based screening. By testing this strategy on the proof-of-principle level with a library of 13 fluorinated monosaccharides to a set of three carbohydrate receptors of diverse origin, i.e. the human macrophage galactose-type lectin, a plant lectin, Pisum sativum agglutinin, and the bacterial Gal-/Glc-binding protein from Escherichia coli, it became possible to simultaneously define their monosaccharide selectivity and identify the essential hydroxyls for interaction.

S-Glycosides: synthesis of S-linked arabinoxylan oligosaccharides.

S-Glycosides are important tools for the elucidation of specific protein-carbohydrate interactions and can significantly aid structural and functional studies of carbohydrate-active enzymes, as they are often inert or act as enzyme inhibitors. In this context, this work focuses on the introduction of an S-linkage into arabinoxylan oligosaccharides (AXs) in order to obtain a small collection of synthetic tools for the study of AXs degrading enzymes. The key step for the introduction of the S-glycosidic linkage involved anomeric thiol S-alkylation of an orthogonally protected l-arabinopyranoside triflate. The resulting S-linked disaccharide was subsequently employed in a series of glycosylation reactions to obtain a selectively protected tetrasaccharide. This could be further elaborated through chemoselective deprotection and glycosylation reactions to introduce branching l-arabinofuranosides.

Unraveling Sugar Binding Modes to DC-SIGN by Employing Fluorinated Carbohydrates.

A fluorine nuclear magnetic resonance (19F-NMR)-based method is employed to assess the binding preferences and interaction details of a library of synthetic fluorinated monosaccharides towards dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), a lectin of biomedical interest, which is involved in different viral infections, including HIV and Ebola, and is able to recognize a variety of self- and non-self-glycans. The strategy employed allows not only screening of a mixture of compounds, but also obtaining valuable information on the specific sugar-protein interactions. The analysis of the data demonstrates that monosaccharides Fuc, Man, Glc, and Gal are able to bind DC-SIGN, although with decreasing affinity. Moreover, a new binding mode between Man moieties and DC-SIGN, which might have biological implications, is also detected for the first time. The combination of the 19F with standard proton saturation transfer difference (1H-STD-NMR) data, assisted by molecular dynamics (MD) simulations, permits us to successfully define this new binding epitope, where Man coordinates a Ca2+ ion of the lectin carbohydrate recognition domain (CRD) through the axial OH-2 and equatorial OH-3 groups, thus mimicking the Fuc/DC-SIGN binding architecture.

Synthesis of lactosamine-based building blocks on a practical scale and investigations of their assembly for the preparation of 19F-labelled LacNAc oligomers.

The ubiquitous disaccharide N-acetyllactosamine (LacNAc type 2, Galß1,4GlcNAc) is often over-expressed on the surface of cancer cells where it is bound by tumour secreted galectins contributing to cancer-related processes such as metastasis, adhesion, tumour survival, and immune escape. To facilitate NMR investigations into the binding interactions between oligo-LacNAc structures and galectins, which can show both exo- and endo-binding behaviour, a library of regioselectively 19F-labelled oligo-LacNAc structures was required. Herein, the synthesis on a practical scale of various N-protected (Troc, Phth, TFAc) lactosamine donors is reported starting from commercially available lactosamine hydrochloride. Investigations into their glycosylations with lactosamine acceptors to form 19F-containing LacNAc oligomers showed that benzylated acceptors significantly improved the yields over acetylated ones, and that, gratifyingly, the almost untried N-trifluoroacetamide (NTFAc) protected donors, already containing the desired 19F-label, were found to be optimal, both considering reaction yields and purification of the glycosylation reactions. The NTFAc group of reducing end acceptors was introduced through N-amide transacylation of linker-equipped LacNAc structures. A [2 + 2] synthetic approach was optimized for the preparation of tetrasaccharide LacNAc/TFAc-dimers and also further expanded to the synthesis of hexasaccharide LacNAc/TFAc-trimer structures.