Tuberculosis (TB) is among the leading causes of death from a single infectious agent i.e., Mycobacterium tuberculosis, resulting in 1.3 million deaths in 2020 (1). The only currently licensed vaccine for TB, the live-attenuated Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine, fails to prevent Mtb infection or protect against pulmonary TB in adults. Consequently, there is a clear need for the development of new vaccines against TB. Here we describe strategies to produce antigens of Mtb for use as novel vaccine candidates.
Mycobacterial PE/PPE proteins account for up to 10% of the coding capacity of the Mtb genome and have a known link to pathogenicity (2). Production of PPE proteins is hampered by their low solubility when expressed as single recombinant E. coli proteins. An IPTG inducible dual expression vector was employed for the simultaneous expression of a ppe gene alongside its cognate pe partner. PPE19 is demonstrated in our lab to be implicated in virulence, however its PE partner remains unconfirmed. Two leading candidates, PE13 or PE31, were co-expressed alongside PPE19. Production was validated via expression in E. coli BL21(DE3) where all proteins were observed via SDS-PAGE and immunoblotting. Validation of the system in an IPTG inducible strain of mycobacteria is underway.
The Mycobacterial membrane protein large (MmpL) protein family contains 13 member proteins responsible for the transport of essential Mtb substrates and supports the pathogenicity of Mtb and its capacity to persist within a host (3). MmpL proteins are characterised by their numerous transmembrane domains and presence of large extracellular or periplasmic loops. Here, three synthetic antigens were constructed comprising of the soluble extracellular regions of MmpL (SERoM)-1, MmpL3 and MmpL11 (SERoM-3 and 11 respectively) as potential vaccine candidates. Synthetic antigens were cloned into pET19b and expressed in E. coli BL21(DE3) prior to purification using an N-terminal polyhistidine-tag. The immunogenicity and protective efficacy of purified SERoM antigens will be examined in vivo in an adjuvanted subunit vaccine trial in combination with the licensed oil-in-water emulsion ASO3.