Production and Evaluation of Ag85B:HspX:hFc1 Immunogenicity as an Fc Fusion Recombinant Multi-Stage Vaccine Candidate Against Mycobacterium tuberculosis

An urgent need is to introduce an effective vaccine against Mycobacterium tuberculosis (M.tb) infection. In the present study, a multi-stage M.tb immunodominant Fc gamma 1 fusion protein (Ag85B:HspX:hFc gamma 1) was designed and produced, and the immunogenicity of purified protein was evaluated. This recombinant fusion protein was produced in the Pichia pastoris expression system. The HiTrap-rPA column affinity chromatography purified and confirmed the fusion protein using ELISA and Western blotting methods. The co-localisation assay was used to confirm its proper folding and function. IFN-gamma, IL-12, IL-4, and TGF-beta expression in C57BL/6 mice then evaluated the immunogenicity of the construct in the presence and absence of BCG. After expression optimisation, medium-scale production and the Western blotting test confirmed suitable production of Ag85B:HspX:hFc gamma 1. The co-localisation results on antigen-presenting cells (APCs) showed that Ag85B:HspX:hFc gamma 1 properly folded and bound to hFc gamma RI. This strong co-localisation with its receptor can confirm inducing proper Th1 responses. The in vivo immunisation assay showed no difference in the expression of IL-4 but a substantial increase in the expression of IFN-gamma and IL-12 (P <= 0.02) and a moderate increase in TGF-beta (P = 0.05). In vivo immunisation assay revealed that Th1-inducing pathways have been stimulated, as IFN-gamma and IL-12 strongly, and TGF-beta expression moderately increased in Ag85B:HspX:hFc gamma 1 group and Ag85B:HspX:hFc gamma 1+BCG. Furthermore, the production of IFN-gamma from splenocytes in the Ag85B:HspX:hFc gamma 1 group was enormously higher than in other treatments. Therefore, this Fc fusion protein can make a selective multi-stage delivery system for inducing appropriate Th1 responses and is used as a subunit vaccine alone or in combination with others.