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International audience; β-(1→3)-D-Glucan is an essential component of the fungal cell wall. Mouse monoclonal antibodies (mAbs) against synthetic nona-β-(1→3)-D-glucoside conjugated with bovine serum albumin (BSA) were generated using hybridoma technology. The affinity constants of two selected mAbs, 3G11 and 5H5, measured by a surface plasmon resonance biosensor assay using biotinylated nona-β-(1→3)-D-glucan as the ligand, were approximately 11 nM and 1.9 nM, respectively. The glycoarray, which included a series of synthetic oligosaccharide derivatives representing β-glucans with different lengths of oligo-β-(1→3)-D-glucoside chains, demonstrated that linear tri-, penta- and nonaglucoside, as well as a β-(1→6)-branched octasaccharide, were recognized by mAb 5H5. By contrast, only linear oligo-β-(1→3)-D-glucoside chains that were not shorter than pentaglucosides (but not the branched octaglucoside) were ligands for mAb 3G11. Immunolabelling indicated that 3G11 and 5H5 interact with both yeasts and filamentous fungi, including species from Aspergillus, Candida, Penicillium genera and Saccharomyces cerevisiae, but not bacteria. Both mAbs could inhibit the germination of Aspergillus fumigatus conidia during the initial hours and demonstrated synergy with the antifungal fluconazole in killing C. albicans in vitro. In addition, mAbs 3G11 and 5H5 demonstrated protective activity in in vivo experiments, suggesting that these β-glucan-specific mAbs could be useful in combinatorial antifungal therapy.

International audience; Biotinylated hepta-, nona- and undeca-α-(1 → 3)-d-glucosides representing long oligosaccharides of α-(1 → 3)-d-glucan, one of the major components of the cell walls of the fungal pathogen Aspergillus fumigatus, were synthesized for the first time via a blockwise strategy. Convergent assembly of the α-(1 → 3)-d-glucan chains was achieved by glycosylation with oligoglucoside derivatives bearing 6- O-benzoyl groups. Those groups are capable of remote α-stereocontrolling participation, making them efficient α-directing tools even in the case of large glycosyl donors. Synthetic biotinylated oligoglucosides (and biotinylated derivatives of previously synthesized tri- and penta-α-(1 → 3)-d-glucosides) loaded on streptavidin microtiter plates were shown to be better recognized by anti-α-(1 → 3)-glucan human polyclonal antibodies and to induce higher cytokine responses upon stimulation of human peripheral blood mononuclear cells than their natural counterpart, α-(1 → 3)-d-glucan, immobilized on a conventional microtiter plate. Attachment of the synthetic oligosaccharides equipped with a hydrophilic spacer via the streptavidin-biotin pair allows better spatial presentation and control of the loading compared to the random sorption of natural α-(1 → 3)-glucan. Increase of oligoglucoside length results in their better recognition and enhancement of cytokine production. Thus, using synthetic α-(1 → 3)-glucan oligosaccharides, we developed an assay for the host immune response that is more sensitive than the assay based on native α-(1 → 3)-glucan.

International audience; 3-Aminopropyl α-(1→3)-pentaglucoside, a fragment of α-(1→3)-glucan of the cell wall of Aspergillus fumigatus, has been synthesized in a blockwise approach. The application of mono- and disaccharide N-phenyltrifluoroacetimidates bearing a stereodirecting 6-O-benzoyl group was essential for stereoselective α-glucosylations. In the products, p-methoxyphenyl and levulinoyl groups served as orthogonal protecting groups for the anomeric position and 3-OH group, respectively. Their removal from shared blocks led to donors and acceptors that were used for the synthesis of pentasaccharides. Coupling of free α-(1→3)-pentaglucoside with biotin and bovine serum albumin (BSA) gave glycoconjugate tools for mycological studies. Immunization of mice with the BSA conjugate induced the generation of antibodies that recognize α-(1→3)-glucan on A. fumigatus cell wall and distinguish its morphotypes. This discovery represents a first step to the development of a diagnostic test system and a vaccine to detect and fight this life-threatening pathogen.