Primary characterization of a panel of monoclonal antibodies raised against the fungal pathogen Candida albicans

Abstract

Background: Candida albicans is an opportunistic pathogen responsible for non-life-threatening mucosal infections and is also a major cause of morbidity and mortality in immunosuppressed patients. While Candida infections can be treated with a variety of fungistatic antifungals, mortality rates from systemic candidiasis remain high. This fungi is capable of switching between various morphological forms including hyphae, associated with biofilm formation and tissue invasion, yeast, which is important for dissemination, the mating-competent opaque form and a commensal morphology that was recently observed in the gastro-intestinal tract. Methods: We immunized SJL and A/J mice with UV-killed hyphae, yeast or opaque C. albicans cells and used a ClonePixFL to select hybridoma clones that secrete immunoglobulins. These were then screened for anti-Candida monoclonal antibodies (mAbs) using an In-Cell ELISA assay. To date, we have produced 38 hybridoma cell lines and concentrated our characterization efforts on 23 mAbs that bind to live C. albicans cells. Results: Five mAbs recognize all morphological forms of C. albicans, 16 are hyphal-specific while 2 will only bind to opaque cells. In addition to C. albicans, 3 of the mAbs also recognize other fungal pathogens such as C. glabrata, C. tropicalis and C. dublininensis. All of the mAbs label the cell surface by immunofluorescence microscopy and 10 produce multiple bands or high-molecular weight smears in western blots. In order to narrow down the identity of mAb targets and to determine whether they recognize glycoproteins or glycans, we used the In-Cell ELISA assay to probe mannosylation ( 06och1, 06pmr1, 06mnt1 06mnt2) and chitin synthase ( 06chs2, 06chs3, 06chs8, 06chs2 06chs8) mutants. We have also produced and probed reverse-phase protein microarrays spotted with total protein extracts from 2,357 GRACE C. albicans strains in the hyphal or yeast morphologies. In these strains, one gene copy is inactivated by insertional mutagenesis while the other allele is under the control of a repressible Tet-promoter. Conclusions: These assays allowed us to cluster the members of this mAb panel according to their target specificities and to prioritize them for future investigations aimed at evaluating their potential for therapeutic or diagnostic purposes.NRC publication: Ye