Binding Affinity and Antifungal Activity of Immune-Fusion Proteins against Candida albicans

摘要

Candida albicans is a yeast-like fungal pathogen that can cause infections ranging from superficial to life-threatening systemic candidiasis. Current treatments for systemic candidiasis are available but often ineffective and toxic. Consequently, it is necessary to develop new therapeutic approaches. The purpose of this study was to construct antibody-based fusion proteins that can bind to C. albicans cells and eliminate them. Two such fusion proteins were constructed. Each one is composed of M1 Fab as the antibody component that binds to C. albicans mannan and the antifungal peptide HPRP-A1. HPRP-A1 was attached via a 15-amino acid linker to either the C-terminus of the constant light chain of M1 Fab (M1 Fab-HPRP-CL) or the N-terminus of the variable light chain of M1 Fab (M1 Fab-HPRP-VL). Binding of the fusion proteins to purified C. albicans mannan was assessed with enzyme-linked immunosorbent assay and the half maximal effective concentration (EC50) for each fusion protein was estimated. EC50 for M1 Fab-HPRP-CL was 273.6 compared to 74.1 for the original M1 Fab (p < 0.05), whereas M1 Fab-HPRP-VL did not show any binding activity, indicating a negative impact on the antibody binding by the linked peptide. Similarly, M1 Fab-HPRP-CL also showed reduced binding for C. albicans cells when compared to M1 Fab as determined with immunofluorescence microscopy and flow cytometry. The effect of M1 Fab-HPRP-CL on the growth of C. albicans cells was analysed using microdilution and absorbance. At 16 microM, the growth of yeast cells treated with M1 Fab-HPRP-CL was 47.1 % of the growth control, compared to 43.5 % for M1 Fab (p > 0.05) and to 1.9 % for HPRP-A1 by itself (p < 0.001). Moreover, HPRP-A1 killed C. albicans at 32 microM and 64 microM, while M1 Fab and M1 Fab-HPRP-CL did not, indicating a loss of the antifungal activity of HPRP-A1 when attached to the antibody. These data together provide valuable insights into the development of novel antibody-based therapeutics as an alternative treatment for candidiasis.