Bacteriophage-based Single-Domain Antibodies For The Therapy Of Systemic Infection Caused By C.albicans

Invasive fungal infection is a predominant cause of human mortality worldwide.The frequency of fungal infections continues to increase in concert with the growing immunocompromised patients,individuals infected with HIV as well as those undergoing chemotherapy,major surgery or transplantation of solid organs or hematopoietic stem cells.C.albicans is the most common cause of hospital-acquired infectious disease and the primary cause of systemic candidiasis,with mortality rate approaching 50%.The treatment of systemic C.albicans infection is notoriously challengeable.Only four chemical drugs are currently used in clinical practice to treat systemic fungal infections including fluoropyrimidine analogs,azoles,polyenes,echinocandins.Despite of progress in the development of the treatment for C.albicans infection in recent years,morbidity and mortality rates still remain very high.The frequent emergence of drug resistance in pathogen population asks us to develop new therapeutic strategies for fungal infectious disease.In this study,we obtained a bacteriophage-based single-domain antibody(scFv)which could bind to C.albicans cell wall.It could be a potential candidate for the therapy of systemic infection caused by C.albicans.The main purpose of this study is to screen a cell wall protein specifically binding scFv antibody and evaluate its antifungal effect in vitro and in vivo.The main methods and results were as follows:1.The selection of human single scFv-phage.After four rounds of bio-panning,we obtained five different bacteriophage-based single-domain antibodies which could bind to the cell wall of C.albicans,named JC1,JC2,JC3,JC4,JC5.2.Binding ability analysis.We analyzed the titer of five antibodies,and JC1 showed the best binding ability by using Western blotting detection and immunofluorescence observation.3.Antifungal effect assay of JC1 in vitro.We chose the suitable working concentration by CCK analysis.Subsequently,time-kill curves indicated the best working time and temperature were obtained.We analyzed the C.albicans cell cycle by FCM analysis.The apoptosis and necrosis of C.albicans were determined by Hoechst and PI double-staining method.SEM was employed to confirm if JC1 had effects on C.albicans cell wall.8 hours later,the results showed that 1×109pfu/ml JC1 could inhibit C.albicans cell proliferation at 30? significantly.Furthermore,JC1 could induce cell cycle arrest of C.albicans,promote apoptosis and necrosis,and result in shrinkage of C.albicans cell wall.4.A systemic C.albicans infection model of mice was used to investigate the effect of JC1 on the fungal burden in kidney,spleen or liver,and the effect on the survival time of the mice.The results showed that JC1 could significantly decrease the fungal burden in kidney,spleen and liver,and prolong the survival time of mice.In conclusion,JC1 which combines the advantage of phage and scFv played an efficient therapeutic role in candidiasis.Therefore,the use of JC1 might provide a new platform for the treatment against candidiasis.