The Mechanism And Functional Domain Of A Novel Adjuvant TFPR1

The recombinant protein TFPR1 is a novel adjuvant that has authorized national patent,which is derived from the triflin's N-terminal PR-1 domain,augmenting a Th1-biased Th1 / Th2 antibody responses and cellular immune response by simply mixing with the antigen.However,it's functional domain and mechanism is unclear,and the effectiveness of TFPR1 as a major component of the compound adjuvant needs to be further invesigated.Objective:To study the role of dendritic cells in the function of a novel adjuvant TFPR1,and the functional domain of TFPR1;To detect the feasibility and effectiveness of TFPR1 as a major component of TF-Al,which is a combination of TFPR1 and aluminum.Methods:(1)The role of dendritic cells in the function of TFPR1 acts as an adjuvant Bone marrow cells were collected from four to five week-old male BALB/c mice,and were cultured with complete RPMI 1640 containing rm GM-CSF and rm IL-4 for six days,TFPR1 was added on day 6,and cells were incubated for another 24 hours,LPS was used as positive control,and PBS as negative.The morphology of dendritic cells was observed by ordinary optical microscope and laser confocal microscope,cell surface makers(CD40,CD80,CD86 and MHC?)were detected by flow cytometry,and the cytokines in supernatant were detected by ELISA.(2)Study on the functional domain of TFPR1 The B cell epitopes of TFPR1 were predicted by using online softwares IEDB and homology modeling by using SWISS-MODEL,and the B cell dominant epitopes of TFPR1 were selected by immunizing mice.Then,the functional domain of TFPR1 protein were designed and expressed in E.coli.And the adjuvant activity of the functional domain of TFPR1 were further evaluated with dendritic cells and model antigens.(3)Evaluation of the adjuvant activity of TF-Al which is a combination of TFPR1 and aluminum The adjuvant activity of TF-Al was evaluated by using OVA and recombinant HBs Ag as model antigens.The level of specific antibody Ig G and Ig G1,Ig G2 a in mouse serum was detected by ELISA at a series of dilution at 14 days after the last immunization.The capacity to induce antibody response of TF-Al was compared with TFPR1 and aluminum respectively,and the adjuvant activity and its characteristics of TF-Al were evaluated.Results:(1)TFPR1 can promote dendritic cells maturation and activate cells to produce cytokines Compared with negative control,dendritic cells incubated with TFPR1 for 24 hours were significant different in morphology,but were similar to positive control.Most of the dendritic cells treated with TFPR1 became round and podosomes became shorter,even disappeared,actin distribution changed from two poles of the cell to the membrane,and CD40,CD80,CD86 and MHC?on cell surface up-regulated.ELISA showed dendritic cells treated with TFPR1 secreted high levels of cytokines.These results showed that TFPR1 has the effect of promoting dendritic cells maturation,and activating cells to produce cytokines,and augmenting immune response.(2)The three functional fragments of TFPR1 have different adjuvant activity By deleting the B cell dominant epitopes,TFPR-T,TFPR-hd and TFPR-ta were designed.Compared with full length protein TFPR1,TFPR-T can't enhance OVA immunogenicity and activate DCs to produce cytokines effectively,which is lack of?-helix 1 and ?-sheet 4,indicating that the adjuvant activity of TFPR1 is closely related to its ability to promote cells to produce cytokines.When the ?-helix 1 was complemented at the N-terminus or the ?-sheet 4 was complemented at the C-terminus of TFPR-T,DCs can be efficiently activated to produce cytokines.In addition,the antigenicity of TFPR-ta is significantly lower than the antigenicity of TFPR-hd.(3)TFPR1 can enhance the immunogenicity of the protein antigen and induce Th1-type biased immune responses The adjuvant TF-Al which is a combination of TFPR1 and aluminum,can induce higher levels of the specific antibody Ig G and Ig G1 in mice than that induced by TFPR1 and aluminum alone by simply mixing with the protein antigen(OVA and HBs Ag).Furthermore,compared with induced by TFPR1 and aluminum alone,TF-Al can induce higher levels of Th1 response related antibody Ig G2 a.So,the adjuvant activity of TF-Al is better than that of TFPR1 and aluminum.Conclusion:TFPR1 is capable of promoting dendritic cells maturation,and activating cells to produce cytokines,and augmenting immune response.What's more,the integrity of spatial structure of TFPR1 is necessary for its adjuvant activity,and ?-helix1 and ?-sheet 4 can not be lost at the same time,and TFPR1 can act as an important component of the compound adjuvant.