Construction And Immunogenicity Of The Multi-epitope Protein TP15 Against Mycobacterium Tuberculosis

BackgroudTuberculosis(TB)is an infectious disease caused by Mycobacterium tuberculosis(MTB).Bacillus Calmette-Guerin(BCG)is the only vaccine to prevent TB,but the protective effect of BCG is unstable.Compared with the traditional protein vaccine,Multi-epitope vaccine,which is composed of several optimum epitopes on some target antigens,can overcome the limitations of MHC molecules and be recognized by a variety of genetic backgrounds.Multi-epitope vaccine has a unique advantage with respect to cell-mediated immunity,and can effectively deal with the mutation of pathogenic microorganisms and the unfavorable immune response.Multi-epitope-based TB vaccine provides a new strategy for prophylactic and therapeutic application of TB,however,protein vaccines usually induce weak immunogenicity especial cell-mediated immunity.The adjuvanted Multi-epitope-based TB vaccine is urgent to strengthen Thl-type immune response.In addition,immunization route can affect the Immune effect and the immune effect of maintenance time.Therefore,we need to screen an optimum adjuvant and immunization routes to enhance the protective effect of Multi-epitope-based TB vaccine.MTB is a intracellular bacteria.The Thl-type immune responses play important roles in MTB infection.Therefore,The adjuvanted Multi-epitope-based TB vaccine should induce the Thl-type immune response and strengthen the immune function of macrophages,which is the basis for its further evaluation of a protective immune response.ObjectiveIn order to research the immunogenicity of multi-epitope-based TB vaccine,we should determine the amino acid sequence and the nucleotide sequence of multi-epitope-based protein TP 15(MEP-TP15),and the recombinant MEP-TP15 was obtained through prokaryotic expression system.We compare the immunogenicity of MF59,heat-killed BCG(hBCG)and MF59/hBCG-adjuvanted MEP-TP15 vaccines through subcutaneous and muscular administration in order to evaluate furtherly the protective immune response.Methods(1)Through online epitope prediction software BIMAS,NetCTL 1.2 and SYFPEITHI,the CTL and Th epitopes of eleven MTB membrane proteins were predicted.The candidate synthesized CTL or Th epitopes stimulated the mouse spleen lymphocytes of mice immunized with heat-killed MTB and peripheral blood monouclear cells(PBMCs)of PPD+/PPD-healthy volunteers.ELISA was used to detect the levels of IFN-y in cell culture supernatant.Based on the ability of CTL or Th stimulation cells to secret IFN-y,the dominant CTL and Th epitopes were determined.(2)The order of the tandem optimum CTL and Th epitope peptides was identified by bioinformatics,which combination with linker peptide constructed MEP-TP15.The gene encoding MEP-TP15 was synthetized,and cloned into the prokaryotic expression vector.The recombinant MEP-TP15 was expressed in E.coli induced by IPTG and purified by ion exchange chromatography.SDS-PAGE was used to detect the expression of MEP-TP15 in E.coli,purity and the molecular weight of MEP-TP15.(3)BALB/c mice were immunized MF59,hBCG and MF59/hBCG-adjuvanted MEP-TP15 vaccines through subcutaneous and muscular administration for three times at intervals of two weeks,respectively.The mice were sacrificed two weeks after the last immunization.Indirect ELISA was used to detect MEP-TP15-specific IgG,IgG1 and IgG2a antibodies in serum.The splenic lymphocytes and peritoneal macrophages were isolated and cultured with MEP-TP 15.Flow cytometry was used to detect the percentage of IL-2+,IFN-y+,IL-4+ and IL-10+CD4+ T cells in cultured splenic lymphocytes.Sandwich ELISA was used to detect the levels of IL-1? and IL-12 in the culture supernatants of cultured peritoneal macrophages.Results(1)Through three kinds of online epitope prediction software,thirty-one CTL epitopes and eighteen Th epitopes of eleven MTB membrane proteins were initially predicted.(2)All candidate epitopes could stimulate mouse spleen lymphocytes of mice immunized with heat-killed MTB and PBMCs from PPD+/PPD-healthy volunteers to secrete IFN-y.According to the level of IFN-?,seven optimum CTL and Th epitopes were screened.(3)MEP-TP15 was expressed in E.coli as inclusion bodies,the purity was higher than 95%through DEAE anion exchange chromatography,and the molecular weight was about 25.5KD.(4)The levels of MEP-TP 15-specific IgG,IgG1 and IgG2a antibodies were significantly increased in serum of mice immunized with MF59,hBCG or MF59/hBCG-adjuvanted MEP-TP 15 vaccines by subcutaneous and intramuscular injection,and MF59/hBCG-adjuvanted MEP-TP15 vaccines was better.MEP-TP15 only induced MEP-TP 15-specific IgG and IgG1 antibodies,but not MEP-TP 15-specific IgG2a antibody when it immunized mice.The levels of IgG2a antibody and the ratios of IgG1/IgG2a were significantly higher and lower with subcutaneous administration than with intramuscular administration of MF59,hBCG or MF59/hBCG-adjuvanted MEP-TP 15 vaccines,respectively.Meanwhile,MF59/hBCG-adjuvanted MEP-TP 15 vaccine was best to induce IgG2a antibody.There were no significant difference in the levels of IgG and IgG1 antibodies between mice immunized with MEP-TP15 and PBS by intramuscular injection.The levels of IgG and IgG1 antibodies were significantly higher in sera from mice immunized with MEP-TP 15 than in serum from mice immunized with PBS by subcutaneous injection.(5)The levels of MEP-TP 15-specific IL-1? and IL-12 secreted by peritoneal macrophages significantly increased in mice immunized with MF59 or MF59/hBCG-adjuvanted MEP-TP 15 vaccines by subcutaneous injection,however,hBCG-adjuvanted MEP-TP 15 vaccine only increased the levels of IL-12 secreted by peritoneal macrophages.(6)The percentage of IL-12+CD4+T cell significantly increased in mice immunized with MF59,hBCG or MF59/hBCG-adjuvanted MEP-TP15 vaccines,and MF 5 9/hB C G-adj u vanted MEP-TP15 vaccine immunized mice could still improve the percentage of IFN-y+CD4+T cell.There was no significant difference in the percentage of IL-4+CD4+T cell,but higher in the percentage of IL-10+CD4+T cell,between mice immunized with MEP-TP15 and mice immunized MF59,hBCG or MF59/hBCG-adjuvanted MEP-TP15 vaccines,respectively.ConclusionMEP-TP 15 is obtained by bioinformatics and genetic engineering techniques.MF59,hBCG or MF59/hBCG adjuvant can significantly enhance immunogenicity of MEP-TP 15,in which MF59 is mainly induced Th2-type immune response,however,MF59/hBCG is mainly induced Thl-type immune response and enhance the immune response of macrophages.The immunogenicity of MF59/hBCG-adjuvanted MEP-TP15 vaccine is better with subcutaneous injection than with intramuscular injection.