Application Of Hybrid Phage Displaying Fba1 Epitope On Protection And Detection Of Candidiasis

Candida albicans is a common commensal and opportunistic fungal pathogen in human,which usually parasitic in mucosal system including respiratory tract,intestinal tract and vaginal.It can cause infections that range from superficial candidiasis of the skin to life-threatening systemic candidiasis,especially in low immunity population,such as patients for cancer,AIDS,long-term taking broad-spectrum antibiotics,organ transplantation and so on.Early diagnosis and treatment for candidiasis is difficult because it has no significant clinical symptoms,and is prone to drug resistance,which leads to high mortality and has became the important causes of death in immunocompromised patients such as cancer patients.Clinical data show that candidiasis has become the second most common cause of death from hospital blood infections.An effective way to reduce the mortality of candidiasis is prevention,early diagnosis and early treatment.However,there is no effective prevention and early diagnosis reagents.The detection method of blood culture is low sensitivity and time-consuming which often delays the optimal treatment period.Therefore,it is important to develop a vaccine and early diagnosis reagent for candidiasis.Phage display is a technology that exogenous peptide expresses on the surface of phage particles by modifying the phage genome.It has extensive application in the development of detection reagents,new vaccine and drug preparation,and provides new means and methods for modern medicine.Filamentous bacteriophage is a good carrier of vaccine and diagnostic reagent with many advantages.Firstly,filamentous bacteriophage is non-toxic to eukaryotic organisms such as human and animals.And it can induce strong immune response as a natural macromolecule,so that the adjuvant with larger side effects can be omitted when vaccinated.Secondly,antigen epitopes are fully displayed on the surface of bacteriophages in the form of multiple copies,and maintain the natural conformation,which makes it easier to be contacted and produce strong antigen-antibody reaction.Finally,filamentous bacteriophage is simple to prepare,easy to transform,stable in physical and chemical properties,suitable for mass production,and its production cost and time-consuming are much lower than traditional vaccine and diagnostic reagent.In view of this,we studyed on the prevention and early diagnosis of candidiasis based on the phage display technology as follows:1.Study on prevention of candidiasis using the phage displaying Fba1 epitopeIn this part,an effective epitope of immunodominant antigen Fba1 of Candida albicans was displayed on pIII or pVIII proteins of filamentous phage fd by phage display technology,and two kinds of epitope-displayed phages were constructed.We verified their immunogenicity by Western Blot technology,and explored their protective effect against C.albicans infection by consturcting a mouse model with candidiasis.Rresults showed that both phages displaying Fba1 epitopes on pIII or pVIII could be recognized by Fba1 polyclonal antibody and had immunogenicity.They could also stimulate mice to produce humoral and cellular immune responses,reduce the colonization of fungi in mice kidney,alleviate kidney injury and increase the survival rate of mice.2.Detection of C.albicans-related antibodies in serum of cancer patientsIn this part,we constructed a double-display phage with Fba1 and Hsp90 epitopes.And we detected the serum of cancer patients(candidiasis susceptible population)and healthy people by ELISA in contrast with single-display phages with Fba1 epitope,single-display phage with Hsp90 epitope and Fba1 and Hsp90 recombinant protein.Results showed that there was no significant difference in sensitivity and specificity between single-display phages and recombinant protein,but double-display phages with both Fba1 and Hsp90 antigen epitopes could significantly improve the sensitivity.In addition,a rapid diagnostic method for candidiasis was established by colloidal gold lateral chromatography with Fba1 recombinant protein as capture antigen.The sensitivity and specificity of this method were similar to that of indirect ELISA,but the detection time was greatly shortened.In summary,we constructed a phage displaying the effective epitope of Candida albicans Fba1 based on phage display technology,and proved its application value in the prevention and early diagnosis of candidiasis,which opened up new ideas for the development of new vaccines and diagnostic reagents for fungal infection.