Synthesis of plant-based, self-adjuvanted, dual antigen specific to Mycobacterium tuberculosis as a novel tuberculosis subunit vaccine that elicits immunogenicity in rabbit

The only approved vaccine, Bacillus Calmette Guérin (BCG) used in global tuberculosis (TB) immunization programmes has been very effective in childhood TB but not in adult pulmonary and latent TB. Moreover, the emergence of multi-drug resistance-TB cases demands either to increase efficiency of BCG or replace it with the one with improved efficacy.A novel combination of two most effective secreted protein antigens specific for Mycobacterium tuberculosis (Mtb), ESAT-6 and MPT-64 (but not present in BCG strains) fused with a cholera toxin B subunit (CTB) and tagged with 6xHis was expressed for the first time in Escherichia coli as well as in transgenic cucumber plants developed using Agrobacterium tumefaciens-mediated transformation. The recombinant fusion protein (His6x.CTB-ESAT6-MPT64) expressed in E. coli was purified by a single-step affinity chromatography and used to produce polyclonal antibodies in rabbit. The transgenic cucumber lines were confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcriptase PCR (RT-PCR), real-time PCR (qRT-PCR) and expression of recombinant fusion protein by western blot analysis and its quantification by enzyme-linked immunosorbent assay (ELISA). A maximum value of the fusion protein, 478 ng.g-1 (0.030% of the total soluble protein) was obtained in a transgenic cucumber line. Rabbit immunized orally showed a significant increase in serum IgG levels against the fusion protein as compared to the non-immunized rabbit.Stable expression of Mtb antigens with CTB in edible cucumber plants (whose fruits are eaten raw) in sufficient amount possibly would facilitate development of a safe, affordable and orally delivered self-adjuvanted, novel dual antigen based subunit vaccine against TB.