Synthesis of an Adjuvant-Free Single Polypeptide-Based Tuberculosis Subunit Vaccine that Elicits In Vivo Immunogenicity in Rats.

A novel tuberculosis subunit vaccine specific for Mycobacterium tuberculosis dual antigens, culture filtrate protein-10 (CFP-10) and antigen 85B (Ag85B) conjugated with cholera toxin non-toxic B subunit (CTB), was expressed as a single polypeptide in high amounts and cost-effectively in Escherichia coli. The recovery and purification conditions for the recombinant fusion protein were established. This simple peptide vaccine required no exogenous adjuvant as it contained CTB, a potent immune modulator. The vaccine's physiochemical, structural, and immunological properties were determined using the in-silico tools. It was highly antigenic, non-allergenic, and non-toxic. Its BlastP search with human proteomes excluded the chances of autoimmune reactions. The tertiary structure model (3D) was validated by Ramachandran plot assessment. The 3D structure docking with Toll-like receptors, TLR-1, 2, 4, and 6, showed that the binding affinity between the vaccine peptide and TLRs was high, and their complex was stable, indicating a strong immune response. The in-silico immune simulation revealed the vaccine-induced both innate and adaptive immune responses. In-vivo validation of the immunogenicity of CTB.CFP10.Ag85B in Wistar rats revealed higher activation of IgG immune response compared to either antigen protein. Similar results were also obtained using the C-ImmSim simulation online server. A comparison of immunogenicity of CTB.CFP10.Ag85B with the only available TB vaccine, Bacillus Calmette-Guérin (BCG) or as a booster after vaccination of Wistar rats with BCG, indicated that the IgG levels were the highest in rats vaccinated with BCG, followed by a booster dose of CTB.CFP10.Ag85B fusion protein. The fusion protein would be a safe potential vaccine booster candidate in BCG-primed individuals against TB.