Development And Efficacy Evaluation Of Four Tuberculosis Subunit Vaccine

1. Immunogenicity of Mycobacterium tuberculosis subunit vaccine ESAT6-RpfEObjective: To clone, express and purify Mycobacterium tuberculosis Esat6-rpfE fusion protein and study its immunogenicity.Methods:The genes of esat6and rpfE were amplified by PCR from genomic DNA of Mycobacterium tuberculosis reference strain H37Rv or clinical stain and then inserted into cloning vector pET30a respectively. The constructed recombinant vector was transformed into host Escherichia coli BL21(DE3). The fusion protein ESAT6-RpfE was expressed and purified after two successive chromatographic purification steps. Sample of ESAT6-RpfE was purified on the anon exchange column and hydrophobic chromatography column. A subunit vaccine was formed by mixing the purified protein with the adjuvant of DDA. C57BL/6mice were immunized every three weeks on the inguinal region subcutaneously for three times. Eight weeks after the last immunization, mice were sacrificed, and the level of lymphocyte secrected antigen-specific IFN-γ was determined. And the level of antigen specific IgG2b and IgG1in sera were dectected by ELISA.Results: The recombinant fusion protein ESAT6-RpfE was overexpressed in inclusion bodies. With the stimulation of RpfE, the level of specific IFN-γ from mice immunized was higher then BCG (p<0.05) and saline control(p<0.01).ESAT6-RpfE immunized mice can induce antigen-spcific antibody.Conclusion:The recombinant fusion protein ESAT6-RpfE was successfully constructed and expressed. It can induce the cell mediated immunity and humoral immunity in C57BL/6mice. ESAT6and RpfE fusion protein can be used as novel components of the new TB vaccine for further evaluation.2. Evaluation of Protective efficacy of the TB subunit vaccineObjective:To evaluate the Immunogenicity and protection of four fusion proteins ESAT6-Ag85B, TB10.4-Ag85B, ESAT6-TB8.4, and ESAT6-RpfE, which was combined with adjuvant.Methods:The subunit vaccine was formed by mixing fusion protein (ESAT6-Ag85B, TB10.4-Ag85B, ESAT6-TB8.4, ESAT6-RpfE) with the adjuvant of DPG [DDA plus poly(I:C) plus gelatin]. BCG and PBS were used as control. The subunit vaccines were given three times with a3-week interval. The number of antigen-specific IFN-γ producing cells was analyzed by ELISPOT ten weeks after boosting, followed by the challenge of H37Rv strain. Six weeks after the infection, the load of M. tuberculosis in mice and the histopathology were analyzed. The immune response and protective efficacy of different subunit vaccines with adjuvant were analyzed.Results:Mice were immunized three times with fours subunit vaccines, and produced stronger antigen-specific interferon-γ. The CFU in the lungs of mice vaccinated with four subunit vaccines were less than PBS controls. All mice immunized with the subunit vaccines except ESAT6-Ag85B presented fewer lesions in lungs than PBS controls.Conclusion:The subunit vaccines consisting of four fusion proteins mixing adjuvant, which showed high immunogenicity and protection against virulent Mycobacterium tuberculosis infection.