The formation of fungal biofilms remains a serious medical problem. In particular, once biofilms are formed they display high levels of resistance against most common antifungals.  Candida albicans and Candida dubliniensis are closely related organisms with respect to morphology and phylogeny, but differ in their expression of virulence-associated proteins involved in adherence and biofilm development. Complement receptor 3-related protein (CR3-RP) is one of the key surface antigens expressed by Candida species during biofilm formation. The main objective of this research was to investigate the role of the polyclonal antibody (Ab) directed against antigen CR3-RP in the inhibition of biofilm formation of C. albicans and C. dubliniensis using different in vitro, ex vivo and in vivo models, and to assess a potential effect of Ab treatment on preformed biofilm of both species. For in vitro experiments, biofilms formed in the presence of Ab were studied using the XTT reduction assay for assessing cell metabolic activity and by crystal violet staining. Confocal Laser. Scanning Microscopy (CLSM) was used for quantifying biomass and architecture. In ex vivo experiments, biofilms formed on cryo-cuts from mouse tongues in the presence of Ab were evaluated by PAS staining. The Galleria mellonella infectious model was selected as an in vivo assay to assess survival. Results demonstrated decreased metabolic activity and biomass of biofilms exposed to Ab in both tested Candida species. When antibody was added at the beginning of biofilm formation, the reduction in metabolic activity was 70% and 46% for C. albicans SC5314 and C. dubliniensis CBS7987 respectively, whereas biofilm biomass was reduced by 90% and 53%. CLSM revealed a decreased thickness of biofilms formed by both species in the presence of Ab. The effectiveness of Ab on preformed 24h biofilms of C. albicans and C. dubliniensis was also tested. Results showed a reduction in metabolic activity of biofilm for both species (26 % and 20 % for C. albicans and C. dubliniensis, respectively). Immunohistochemistry of biofilms formed on mouse tongues confirmed the effectiveness of Ab in reducing both Candida species biofilms ex vivo. Additionally, the in vivo assay proved that Ab administration 1h post-infection increased larval survival compared to control group in the G. mellonella infection model of systemic candidiasis, for both C. albicans and C. dubliniensis. Our data suggest a potential activity of the anti-CR3-RP Ab relevant to immunotherapy or vaccine development against biofilm-associated Candida infections.

This work was supported by the project VEGA_1/0628/15; the Slovak Research and Development Agency under the contract No.APVV-15-0347; and by the research grants FEMS-RG-2015-0045. This research was also supported by Fulbright Commission in Slovak Republic (J. CH. was the Fulbright Visiting Student Researcher at J.L.L.R lab during period Sept 2017 – Feb 2018).

Effectiveness of polyclonal antibody anti-CR3-RPAb directed against surface antigen CR3-RP in the biofilm development of Candida albicans and Candida dubliniensis