Date Published: April 25, 2019
Publisher: Public Library of Science
Author(s): Andrey L. Matveev, Vadim B. Krylov, Yana A. Khlusevich, Ivan K. Baykov, Dmitry V. Yashunsky, Ljudmila A. Emelyanova, Yury E. Tsvetkov, Alexander A. Karelin, Alevtina V. Bardashova, Sarah S. W. Wong, Vishukumar Aimanianda, Jean-Paul Latgé, Nina V. Tikunova, Nikolay E. Nifantiev, Joseph J. Barchi.
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.
The incidence of invasive fungal infections continues to increase, and successful treatment of the diseases remains a serious problem despite the development of more effective antifungal preparations with reduced toxicity [1,2]. Early detection of invasive fungal infections is extremely important for successful treatment. Invasive fungal infections in humans are caused mainly by the species from Aspergillus, Candida, Cryptococcus, and Fusarium genera. Structurally, fungal cells are protected by a cell wall composed of different polysaccharides; while establishing infection, this cell wall undergoes modification and rearrangement, during which some fragment of these polysaccharides are expected to be released . One of the major and essential components of the fungal cell wall is β-(1→3)-D-glucan [4–6]. Detection and quantitative evaluation of this polysaccharide is an important challenge for clinical diagnosis, food control, and ecology monitoring. Currently, Glucatell and related kits for measurement of β-(1→3)-D-glucans with a glucan-reactive preparation of Limulus amebocyte lysate (LAL) [7–9] is widely used; however, it shows a high rate of false positive results for fungal infection . Therefore, an antibody-based enzyme immune-assay (EIA) can be regarded as a practical alternative to the LAL-test in many cases, as it is less expensive and can be sufficiently sensitive to detect β-(1→3)-D-glucan in clinical samples . Several EIAs were developed to date based on polyclonal and monoclonal antibodies [11–13] that were obtained against β-glucans and their BSA-conjugates. Their specificity was evaluated with the use of polysaccharide preparations isolated from natural sources, and therefore the tests were insufficiently characterized.