Numerous studies have demonstrated that targeting immunogens to FcγR on antigen-presenting cells (APCs) can selectively uptake and increase cellular immunity in vitro and in vivo. Therefore， the present study was conducted to evaluate immunogenicity of a novel multistage tuberculosis vaccine， a combination of an early and a dormant immunogenic protein， ESAT6 and HspX， fused to Fcγ2a fragment of mouse IgG2a to target all forms of tuberculosis. Codon-optimized genes consisting of ESAT6， a linker， and HspX fused either to mouse Fcγ2a (ESAT6:HspX:mFcγ2a) or 6× His-tag (ESAT6:HspX:His) were synthesized. The resulting proteins were then produced in Pichia pastoris. The fusion proteins were separately... More
Numerous studies have demonstrated that targeting immunogens to FcγR on antigen-presenting cells (APCs) can selectively uptake and increase cellular immunity in vitro and in vivo. Therefore， the present study was conducted to evaluate immunogenicity of a novel multistage tuberculosis vaccine， a combination of an early and a dormant immunogenic protein， ESAT6 and HspX， fused to Fcγ2a fragment of mouse IgG2a to target all forms of tuberculosis. Codon-optimized genes consisting of ESAT6， a linker， and HspX fused either to mouse Fcγ2a (ESAT6:HspX:mFcγ2a) or 6× His-tag (ESAT6:HspX:His) were synthesized. The resulting proteins were then produced in Pichia pastoris. The fusion proteins were separately emulsified in dimethyldioctadecylammonium bromide(DDA)-trehalose-6，6-dibehenate(TDB) adjuvant， and their immunogenicity with and without bacille Calmette-Guérin (BCG) was assessed in C57BL/6 mice. Th1， Th2， Th17， and T-reg cytokine patterns were evaluated using the ELISA method. Both multistage vaccines induced very strong IL-12 and IFN-γ secretion from splenic cells; the Fc-tagged subunit vaccine induced a more effective Th1 immune response (IFN-γ， 910 pg/mL， and IL-12， 854 pg/mL) with a very low increase in IL-17 (∼0.1 pg/mL) and IL-4 (37 pg/mL) and a mild increase in TGF-β (543 pg/mL) compared to the BCG or ESAT6:HspX:His primed and boosted groups. The production of IFN-γ to ESAT6:HspX:Fcγ2a was very consistent and showed an increasing trend for IL-12 compared to the BCG or ESAT6:HspX:His primed and boosted groups. Fcγ2a used as a delivery vehicle supported the idea of selective uptake， inducing cross-presentation and forming a proper anti-tuberculosis response in context of Th1/Th2 and Th17/T-reg balances， which is important for protection and prevention of damage.
