Using transposon mutagenesis of mycobacterium bovis BCG to identify candidate molecules for novel control approaches for bovine tuberculosis.

Bovine tuberculosis is of huge animal health and economic concern in the UK. Current ‘test and slaughter’ control measures are not sufficient at controlling the disease, with evidence suggesting the introduction of an efficacious cattle vaccine being a viable alternative to reduce annual herd incidence. Approaches using the human tuberculosis vaccine, the live attenuated Mycobacterium bovis strain Bacillus Calmette–Guérin (BCG) Danish, as part of a prime/boost strategy have been shown to be effective at inducing immunity in cattle. One of the most effective vaccination strategies involves the development of a subunit vaccine for use alongside BCG. To this end, identification of protective antigens for use in a subunit vaccine is key. Here we describe a BCG transposon library mutant selection in cattle to identify the protective antigens of BCG. Prior to the cattle screen, the ‘selectability’ of the transposon library was validated using the mycobacterial heat-shock response and isoniazid resistance mechanisms. The preliminary screens identified essential genes in the respective pathways, such as hspR and katG, validating the use of the library to select for genes relevant to a selection pressure. Following validation, BCG vaccinated and immunologically naïve cattle were intranodally challenged with the transposon library and transposon mutants with comparatively better survival in BCG vaccinated animals were identified using TnSeq. The screen identified Mb0025, Mb0086, YrbE1B, PPE20 and Mb2235 as potential antigenic proteins of BCG. Mb0025 and Mb2235 were recombinantly expressed using an E. coli expression system. Mb0086, YrbE1B and PPE20 were purchased as peptides. The immunogenicity of the selected proteins was assessed in PBMCs harvested from immunologically naïve and BCG vaccinated cattle. IFN-γ, IP10, IL-β, IL-17a, IL-10 and IL-12 release from simulated PBMCs was quantified. IP10 production significantly increased upon stimulation with Mb0086, YrbE1B and PPE20 in vaccinated animals, with a trend of increased IL-17a and IL-10. Mb0086 and YrbE1B stimulated an increase in IL-1β production. Although protection has not been proven, this study has acted as a gate to select the best possible candidates for further experimentation in small animal studies. The transposon library screen also identified genes encoding enzymes involved in the synthesis of glycolipid antigens, such as ppm1 and ltp2, suggesting the inclusion of immunogenic glycolipids in a subunit vaccine along with protein antigens as an area for further study